Nematode fatty acid desaturase-like sequences

ABSTRACT

Nucleic acid molecules from nematodes encoding fatty acid desaturase polypeptides are described. Fatty acid desaturase-like polypeptide sequences are also provided, as are vectors, host cells, and recombinant methods for production of fatty acid desaturase-like nucleotides and polypeptides. Also described are screening methods for identifying inhibitors and/or activators of fatty acid desaturase-like polypeptides, as well as methods for antibody production.

RELATED APPLICATION INFORMATION

[0001] This application claims priority to provisional applicationserial No. 60/322,003, filed Sep. 13, 2001.

BACKGROUND

[0002] Nematodes (derived from the Greek word for thread) are active,flexible, elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. While only 20,000 species of nematode have beenidentified, it is estimated that 40,000 to 10 million actually exist.Some species of nematodes have evolved as very successful parasites ofboth plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans (Whitehead (1998) Plant Nematode Control. CAB International, NewYork).

[0003] Nematode parasites of plants can inhabit all parts of plants,including the roots, developing flower buds, leaves, and stems. Plantparasites are classified on the basis of their feeding habits into thebroad categories: migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites and establish long-term infectionswithin roots that are often very damaging to crops (Whitehead, supra).It is estimated that parasitic nematodes cost the horticulture andagriculture industries in excess of $78 billion worldwide a year, basedon an estimated average 12% annual loss spread across all major crops.For example, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al. (1994)Plant and Soil Nematodes: Societal Impact and Focus for the Future. TheCommittee on National Needs and Priorities in Nematology. CooperativeState Research Service, US Department of Agriculture and Society ofNematologists). Several factors make the need for safe and effectivenematode controls urgent. Continuing population growth, famines, andenvironmental degradation have heightened concern for the sustainabilityof agriculture, and new government regulations may prevent or severelyrestrict the use of many available agricultural anthelmintic agents.

[0004] The situation is particularly dire for high value crops such asstrawberries and tomatoes where chemicals have been used extensively tocontrol soil pests. The soil fumigant methyl bromide has been usedeffectively to reduce nematode infestations in a variety of thesespecialty crops. It is however regulated under the U.N. MontrealProtocol as an ozone-depleting substance and is scheduled forelimination in 2005 in the United States (Carter (2001) CalifoniaAgriculture, 55(3):2). It is expected that strawberry and othercommodity crop industries will be significantly impacted if a suitablereplacement for methyl bromide is not found. Presently there are a verysmall array of chemicals available to control nematodes and they arefrequently inadequate, unsuitable, or too costly for some crops or soils(Becker (1999) Agricultural Research Magazine 47(3):22-24; U.S. Pat. No.6,048,714). The few available broad-spectrum nematicides such as Telone(a mixture of 1,3-dichloropropene and chloropicrin) have significantrestrictions on their use because of toxicological concerns (Carter(2001) California Agriculture, Vol. 55(3):12-18).

[0005] Fatty acids are a class of natural compounds that have beeninvestigated as alternatives to the toxic, non-specific organophosphate,carbamate and fumigant pesticides (Stadler et al. (1994) Planta Medica60(2):128-132; U.S. Pat. Nos. 5,192,546; 5,346,698; 5,674,897;5,698,592; 6,124,359). It has been suggested that fatty acids derivetheir pesticidal effects by adversely interfering with the nematodecuticle or hypodermis via a detergent (solubilization) effect, orthrough direct interaction of the fatty acids and the lipophilic regionsof target plasma membranes (Davis et al. (1997) Journal of Nematology29(4S):677-684). In view of this general mode of action it is notsurprising that fatty acids are used in a variety of pesticidalapplications including as herbicides (e.g., SCYTHE by Dow Agrosciencesis the C9 saturated fatty acid pelargonic acid), as bactericides andfungicides (U.S. Pat. Nos. 4,771,571; 5,246,716) and as insecticides(e.g., SAFER INSECTICIDAL SOAP by Safer, Inc.).

[0006] The phytotoxicity of fatty acids has been a major constraint ontheir general use in agricultural applications (U.S. Pat. No. 5,093,124)and the mitigation of these undesirable effects while preservingpesticidal activity is a major area of research. The esterification offatty acids can significantly decrease their phytotoxicity (U.S. Pat.Nos. 5,674,897; 5,698,592; 6,124,359). Such modifications can howeverlead to dramatic loss of nematicidal activity as is seen for linoleic,linolenic and oleic acid (Stadler et al. (1994) Planta Medica60(2):128-132) and it may be impossible to completely decouple thephytotoxicity and nematicidal activity of pesticidal fatty acids becauseof their non-specific mode of action. Perhaps not surprisingly, thenematicidal fatty acid pelargonic acid methyl ester (U.S. Pat. Nos.5,674,897; 5,698,592; 6,124,359) shows a relatively small “therapeuticwindow” between the onset of pesticidal activity and the observation ofsignificant phytotoxicity (Davis et al. (1997) J Nematol29(4S):677-684). This is the expected result if both the phytotoxicityand the nematicidial activity derive from the non-specific disruption ofplasma membrane integrity. Similarly the rapid onset of pesticidalactivity seen with many nematicidal fatty acids at therapeuticconcentrations (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) suggestsa non-specific mechanism of action, possibly related to the disruptionof membranes, action potentials and neuronal activity.

[0007] Ricinoleic acid, the major component of castor oil, providesanother example of the unexpected effects esterification can have onfatty acid activity. Ricinoleic acid has been shown to have aninhibitory effect on water and electrolyte absorption using evertedhamster jejunal and ileal segments (Gaginella et al. (1975) J PharmacolExp Ther 195(2):355-61) and to be cytotoxic to isolated intestinalepithelial cells (Gaginella et al. (1977) J Pharmacol Exp Ther201(1):259-66). These features are likely the source of the laxativeproperties of castor oil, which is given as a purgative in humans andlivestock, e.g., as a deworming agent. In contrast, the methyl ester ofricinoleic acid is ineffective at suppressing water absorption in thehamster model (Gaginella et al. (1975) J Pharmacol Exp Ther195(2):355-61).

[0008] The macrocyclic lactones (e.g., avermectins and milbemycins) anddelta-toxins from Bacillus thuringiensis (Bt) are chemicals that inprinciple provide excellent specificity and efficacy and should allowenvironmentally safe control of plant parasitic nematodes.Unfortunately, in practice, these two approaches have proven lesseffective for agricultural applications against root pathogens. Althoughcertain avermectins show exquisite activity against plant parasiticnematodes these chemicals are hampered by poor bioavailability due totheir light sensitivity, degradation by soil microorganisms and tightbinding to soil particles (Lasota & Dybas (1990) Acta Leiden59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In:The Physiology and Biochemistry of Free-Living and Plant-parasiticNematodes (eds R. N. Perry & D. J. Wright), CAB International 1998).Consequently, despite years of research and extensive use against animalparasitic nematodes, mites and insects (plant and animal applications),macrocyclic lactones (e.g., avermectins and milbemycins) have never beencommercially developed to control plant parasitic nematodes in the soil.

[0009] Bt delta-toxins must be ingested to affect their target organ,the brush border of midgut epithelial cells (Marroquin et al. (2000)Genetics. 155(4):1693-1699). Consequently they are not anticipated to beeffective against the dispersal, non-feeding, juvenile stages of plantparasitic nematodes in the field. These juvenile stages only commencefeeding when a susceptible host has been infected. Thus, Bt delta-toxinsnematicides may need to penetrate the cuticle in order to be effective.In addition, soil mobility of a relatively large 65-130 kDa protein—thesize of typical Bt delta-toxins—is expected to be poor and delivery inplanta is likely to be constrained by the exclusion of large particlesby the feeding tube of certain plant parasitic nematodes such asHeterodera (Atkinson et al. (1998) Engineering resistance toplant-parasitic nematodes. In: The Physiology and Biochemistry ofFree-Living and Plant-parasitic Nematodes (eds R. N. Perry & D. J.Wright), CAB International 1998).

[0010] Many plant species are known to be highly resistant to nematodes.The best documented of these include marigolds (Tagetes spp.), rattlebox(Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.), castorbean (Ricinus communis), margosa (Azardiracta indica), and many membersof the family Asteraceae (family Compositae) (Hackney & Dickerson.(1975) J Nematol 7(1):84-90). The active principle(s) for thisnematicidal activity has not been discovered in all of these examplesand no plant-derived products are sold commercially for control ofnematodes. In the case of the Asteraceae, the photodynamic compoundalpha-terthienyl has been shown to account for the strong nematicidalactivity of the roots. Castor beans are plowed under as a green manurebefore a seed crop is set. However, a significant drawback of the castorplant is that the seed contains toxic compounds (such as ricin) that cankill humans, pets, and livestock and is also highly allergenic.

[0011] There remains an urgent need to develop environmentally safe,target-specific ways of controlling plant parasitic nematodes. In thespecialty crop markets, economic hardship resulting from nematodeinfestation is highest in strawberries, bananas, and other high valuevegetables and fruits. In the high-acreage crop markets, nematode damageis greatest in soybeans and cotton. There are however, dozens ofadditional crops that suffer from nematode infestation including potato,pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals andgolf course turf grasses.

[0012] Nematode parasites of vertebrates (e.g., humans, livestock andcompanion animals) include gut roundworms, hookworms, pinworms,whipworms, and filarial worms. They can be transmitted in a variety ofways, including by water contamination, skin penetration, bitinginsects, or by ingestion of contaminated food.

[0013] In domesticated animals, nematode control or “de-worming” isessential to the economic viability of livestock producers and is anecessary part of veterinary care of companion animals. Parasiticnematodes cause mortality in animals (e.g., heartworm in dogs and cats)and morbidity as a result of the parasites' inhibiting the ability ofthe infected animal to absorb nutrients. Parasite-induced nutrientdeficiency results in diseased livestock and companion animals (i.e.,pets), as well as in stunted growth. For instance, in cattle and dairyherds, a single untreated infection with the brown stomach worm canpermanently stunt an animal's ability to effectively convert feed intomuscle mass or milk.

[0014] Two factors contribute to the need for novel anthelmintics andvaccines for control of parasitic nematodes of animals. First, some ofthe more prevalent species of parasitic nematodes of livestock arebuilding resistance to the anthelmintic drugs available currently,meaning that these products will eventually lose their efficacy. Thesedevelopments are not surprising because few effective anthelmintic drugsare available and most have been used continuously. Presently a numberof parasitic species has developed resistance to most of theanthelmintics (Geents et al. (1997) Parasitology Today 13:149-151;Prichard (1994) Veterinary Parasitology 54:259-268). The fact that manyof the anthelmintic drugs have similar modes of action complicatesmatters, as the loss of sensitivity of the parasite to one drug is oftenaccompanied by side resistance—that is, resistance to other drugs in thesame class (Sangster & Gill (1999) Parasitology Today Vol.15(4):141-146). Secondly, there are some issues with toxicity for themajor compounds currently available.

[0015] Human infections by nematodes result in significant mortality andmorbidity, especially in tropical regions of Africa, Asia, and theAmericas. The World Health Organization estimates 2.9 billion people areinfected with parasitic nematodes. While mortality is rare in proportionto total infections (180,000 deaths annually), morbidity is tremendousand rivals tuberculosis and malaria in disability adjusted life yearmeasurements. Examples of human parasitic nematodes include hookworm,filarial worms, and pinworms. Hookworm is the major cause of anemia inmillions of children, resulting in growth retardation and impairedcognitive development. Filarial worm species invade the lymphatics,resulting in permanently swollen and deformed limbs (elephantiasis) andinvade the eyes causing African Riverblindness. Ascaris lumbricoides,the large gut roundworm infects more than one billion people worldwideand causes malnutrition and obstructive bowl disease. In developedcountries, pinworms are common and often transmitted through children indaycare.

[0016] Even in asymptomatic parasitic infections, nematodes can stilldeprive the host of valuable nutrients and increase the ability of otherorganisms to establish secondary infections. In some cases, infectionscan cause debilitating illnesses and can result in anemia, diarrhea,dehydration, loss of appetite, or death.

[0017] While public health measures have nearly eliminated one tropicalnematode (the water-borne Guinea worm), cases of other worm infectionshave actually increased in recent decades. In these cases, drugintervention provided through foreign donations or purchased by thosewho can afford it remains the major means of control. Because of thehigh rates of reinfection after drug therapy, vaccines remain the besthope for worm control in humans. There are currently no vaccinesavailable.

[0018] Until safe and effective vaccines are discovered to preventparasitic nematode infections, anthelmintic drugs will continue to beused to control and treat nematode parasitic infections in both humansand domestic animals. Finding effective compounds against parasiticnematodes has been complicated by the fact that the parasites have notbeen amenable to culturing in the laboratory. Parasitic nematodes areoften obligate parasites (i.e., they can only survive in theirrespective hosts, such as in plants, animals, and/or humans) with slowgeneration times. Thus, they are difficult to grow under artificialconditions, making genetic and molecular experimentation difficult orimpossible. To circumvent these limitations, scientists have usedCaenorhabidits elegans as a model system for parasitic nematodediscovery efforts.

[0019]C. elegans is a small free-living bacteriovorous nematode that formany years has served as an important model system for multicellularanimals (Burglin (1998) Int. J. Parasitol., 28(3): 395-411). The genomeof C. elegans has been completely sequenced and the nematode shares manygeneral developmental and basic cellular processes with vertebrates(Ruvkin et al. (1998) Science 282: 2033-41). This, together with itsshort generation time and ease of culturing, has made it a model systemof choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med. 32:23-30).

[0020] Although C. elegans serves as a good model system forvertebrates, it is an even better model for study of parasiticnematodes, as C. elegans and other nematodes share unique biologicalprocesses not found in vertebrates. For example, unlike vertebrates,nematodes produce and use chitin, have gap junctions comprised ofinnexin rather than connexin and contain glutamate-gated chloridechannels rather than glycine-gated chloride channels (Bargmann (1998)Science 282: 2028-33). The latter property is of particular relevancegiven that the avermectin class of drugs is thought to act atglutamate-gated chloride receptors and is highly selective forinvertebrates (Martin (1997) Vet. J. 154:11-34).

[0021] A subset of the genes involved in nematode specific processeswill be conserved in nematodes and absent or significantly diverged fromhomologues in other phyla. In other words, it is expected that at leastsome of the genes associated with functions unique to nematodes willhave restricted phylogenetic distributions. The completion of the C.elegans genome project and the growing database of expressed sequencetags (ESTs) from numerous nematodes facilitate identification of these“nematode specific” genes. In addition, conserved genes involved innematode-specific processes are expected to retain the same or verysimilar functions in different nematodes. This functional equivalencehas been demonstrated in some cases by transforming C. elegans withhomologous genes from other nematodes (Kwa et al. (1995) J. Mol. Biol.246:500-10; Redmond et al. (2001) Mol. Biochem. Parasitol. 112:125-131).This sort of data transfer has been shown in cross phyla comparisons forconserved genes and is expected to be more robust among species within aphylum. Consequently, C. elegans and other free-living nematode speciesare likely excellent surrogates for parasitic nematodes with respect toconserved nematode processes.

[0022] Many expressed genes in C. elegans and certain genes in otherfree-living nematodes can be “knocked out” genetically by a processreferred to as RNA interference (RNAi), a technique that provides apowerful experimental tool for the study of gene function in nematodes(Fire et al. (1998) Nature 391(6669):806-811; Montgomery et al. (1998)Proc. Natl. Acad Sci USA 95(26):15502-15507). Treatment of a nematodewith double-stranded RNA of a selected gene can destroy expressedsequences corresponding to the selected gene thus reducing expression ofthe corresponding protein. By preventing the translation of specificproteins, their functional significance and essentiality to the nematodecan be assessed. Determination of essential genes and theircorresponding proteins using C. elegans as a model system will assist inthe rational design of anti-parasitic nematode control products.

SUMMARY

[0023] The invention features nucleic acid molecules encodingMeloidogyne incognita, Heterodera glycines, Dirofilaria immitis,Strongyloides stercoralis and Rhabditella axei fatty acid desaturasesand other nematode fatty acid desaturase-like proteins. M. incognita isa Root Knot Nematode that causes substantial damage to several crops,including cotton, tobacco, pepper, and tomato. H. glycines, referred toas Soybean Cyst Nematode, is a major pest of soybean. D. immitis (dogheartworm) and S. stercoralis (human threadworm) are mammalianparasites. R. axei is a free-living nematode that serves as a model forparasitic nematodes. In part, the fatty acid desaturase-like nucleicacids and polypeptides of the invention allow for the identification ofa nematode species, and for the identification of compounds that bind toor alter the activity of fatty acid desaturase-like polypeptides. Suchcompounds may provide a means for combating diseases and infestationscaused by nematodes, particularly those caused by M. incognita (e.g., intobacco, cotton, pepper, or tomato plants), H. glycines (e.g., insoybeans), D. immitis (e.g., in dogs) and S. stercoralis (e.g., inhumans).

[0024] The invention is based, in part, on the identification of cDNAsencoding M. incognita fatty acid desaturases (SEQ ID NO: 1, SEQ ID NO: 2and SEQ ID NO: 3). These 1194, 1242 and 1260 nucleotide cDNAs have 1191,1239 and 1161 nucleotide open reading frames encoding 397, 413 and 387amino acid polypeptides (SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10)respectively.

[0025] The invention is also based, in part, on the identification of acDNA encoding H. glycines fatty acid desaturase (SEQ ID NO: 4). This1488 nucleotide cDNA has a 1167 nucleotide open reading frame encoding a389 amino acid polypeptide (SEQ ID NO: 11).

[0026] The invention is also based, in part, on the identification of apartial cDNA fragment encoding D. immitis fatty acid desaturase (SEQ IDNO: 5). This 1068 nucleotide cDNA has a 867 nucleotide open readingframe encoding a 289 amino acid polypeptide (SEQ ID NO: 12).

[0027] The invention is also based, in part, on the identification of acDNA encoding S. stercoralis fatty acid desaturase (SEQ ID NO: 6). This1221 nucleotide cDNA has a 1104 nucleotide open reading frame encoding a368 amino acid polypeptide (SEQ ID NO: 13).

[0028] The invention is also based, in part, on the identification of acDNA encoding R. axei fatty acid desaturase (SEQ ID NO: 7). This 1233nucleotide cDNA has a 1122 nucleotide open reading frame encoding a 374amino acid polypeptide (SEQ ID NO: 14).

[0029] In one aspect, the invention features novel nematode fatty aciddesaturase-like polypeptides. Such polypeptides include purifiedpolypeptides having the amino acid sequences set forth in SEQ ID NO: 8,9, 10, 11, 12, 13 and 14. Also included are polypeptides having an aminoacid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or98% identical to SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. The purifiedpolypeptides can be encoded by a nematode gene, e.g., a nematode geneother than a C. elegans gene. For example, the purified polypeptide hasa sequence other than SEQ ID NO: 32 (C. elegans fatty acid desaturase).The purified polypeptides can further include a heterologous amino acidsequence, e.g., an amino-terminal or carboxy-terminal sequence. Alsofeatured are purified polypeptide fragments of the aforementioned fattyacid desaturase-like polypeptides, e.g., a fragment of at least about20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 325, 350, 375, 395,400 amino acids. Non-limiting examples of such fragments include:fragments from about amino acid 1 to 50, 1 to 75, 1 to 92, 1 to 96, 1 to100, 1 to 125, 1 to 399, 51 to 100, 92 to 150, 96 to 150, 92 to 400, 200to 300, and 1 to 380 of SEQ ID NO: 8, 9, 10, 11, 12, 13 and 14. Thepolypeptide or fragment thereof can be modified, e.g., processed,truncated, modified (e.g. by glycosylation, phosphorylation,acetylation, myristylation, prenylation, palmitoylation, amidation,addition of glycerophosphatidyl inositol), or any combination of theabove. These various polypeptide fragments can be used for a variety ofpurposes, including for the eliciting of antibodies directed against afatty acid desaturase-like polypeptide.

[0030] In another aspect, the invention features novel isolated nucleicacid molecules encoding nematode fatty acid desaturase-likepolypeptides. Such isolated nucleic acid molecules include nucleic acidscomprising, consisting of or consisting essentially of the nucleotidesequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7. Also includedare isolated nucleic acid molecules having the same sequence as orencoding the same polypeptide as a nematode fatty acid desaturase-likegene (other than C. elegans fatty acid desaturase-like genes).

[0031] Also featured are: 1) isolated nucleic acid molecules having astrand that hybridizes under low stringency conditions to a singlestranded probe of the sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 ortheir complements and, optionally, encodes polypeptides of between 375and 425 amino acids and preferably have Δ12 fatty acid desaturaseactivity; 2) isolated nucleic acid molecules having a strand thathybridizes under high stringency conditions to a single stranded probeof the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 or theircomplements and, optionally, encodes polypeptides of between 375 and 425amino acids and preferably have A 12 fatty acid desaturase activity; 3)isolated nucleic acid fragments of a fatty acid desaturase-like nucleicacid molecule, e.g., a fragment of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 thatis about 230, 435, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1450or more nucleotides in length or ranges between such lengths; and 4)oligonucleotides that are complementary to a fatty acid desaturase-likenucleic acid molecule or a fatty acid desaturase-like nucleic acidcomplement, e.g., an oligonucleotide of about 10, 15, 18, 20, 22, 24,28, 30, 35, 40, 50, 60, 70, 80, or more nucleotides in length. Exemplaryoligonucleotides are oligonucleotides which anneal to a site locatedbetween nucleotides about 1 to 24, 1 to 48, 1 to 60, 1 to 120, 24 to 48,24 to 60, 49 to 60, 61 to 180, 145 to 165, 165 to 185, 1260 to 1280,1281 to 1300, 1301 to 1320, 1321 to 1340, 1341 to 1360, 1361 to 1380,1381 to 1400, 1401 to 1420, 1421 to 1456 of SEQ ID NO: 1, 2, 3, 4, 5, 6or 7. Such nucleic acid fragments are useful for detecting the presenceof fatty acid desaturase-like mRNA. Nucleic acid fragments include thefollowing non-limiting examples: nucleotides about 1 to 200, 100 to 300,200 to 400, 300 to 500, 400 to 600, 500 to 700, 600 to 800, 700 to 900,800 to 1000, 900 to 1100, 1000 to 1200, 1100 to 1300, 1200 to 1400, 1300to 1456 of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7. Also within the inventionare nucleic acid molecules that hybridize under stringent conditions tonucleic acid molecules comprising SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 andcomprise 3,000, 2,000, 1,000 or fewer nucleotides and preferably encodea polypeptide having Δ12 fatty acid desaturase activity and/or have asequence corresponding to that of a naturally occurring nematode. Theisolated nucleic acid can further include a heterologous promoteroperably linked to the fatty acid desaturase-like nucleic acid molecule.

[0032] A molecule featured herein can be from a nematode of the classAraeolaimida, Ascaridida, Chromadorida, Desmodorida, Diplogasterida,Monhysterida, Mononchida, Oxyurida, Rhigonematida, Spirurida, Enoplia,Desmoscolecidae, Rhabditida, or Tylenchida. Alternatively, the moleculecan be from a species of the class Rhabditida, particularly a speciesother than C. elegans.

[0033] In another aspect, the invention features a vector, e.g., avector containing an aforementioned nucleic acid. The vector can furtherinclude one or more regulatory elements, e.g., a heterologous promoter.The regulatory elements can be operably linked to the fatty aciddesaturase-like nucleic acid molecules in order to express a fatty aciddesaturase-like nucleic acid molecule. In yet another aspect, theinvention features a transgenic cell or transgenic organism having inits genome a transgene containing an aforementioned fatty aciddesaturase-like nucleic acid molecule and a heterologous nucleic acid,e.g., a heterologous promoter.

[0034] In still another aspect, the invention features an antibody,e.g., an antibody, antibody fragment, or derivative thereof that bindsspecifically to an aforementioned polypeptide. Such antibodies can bepolyclonal or monoclonal antibodies. The antibodies can be modified,e.g., humanized, rearranged as a single-chain, or CDR-grafted. Theantibodies may be directed against a fragment, a peptide, or adiscontinuous epitope from a fatty acid desaturase-like polypeptide.

[0035] In another aspect, the invention features a method of screeningfor a compound that binds to a nematode fatty acid desaturase-likepolypeptide, e.g., an aforementioned polypeptide. The method includesproviding the nematode polypeptide; contacting a test compound to thepolypeptide; and detecting binding of the test compound to the nematodepolypeptide. In one embodiment, the method further includes contactingthe test compound to a mammalian or plant fatty acid desaturase-likepolypeptide; and detecting binding of the test compound to the mammalianor plant fatty acid desaturase-like polypeptide. A test compound thatbinds the nematode fatty acid desaturase-like polypeptide with at least2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold affinity greaterrelative to its affinity for the mammalian (e.g., a human) or plantfatty acid desaturase-like polypeptide can be identified.

[0036] Alternatively, the compounds can bind to the mammalian or plantfatty acid desaturase with affinity similar to that of the nematodefatty acid desaturase, but not be significantly detrimental to a plantand/or animal.

[0037] The invention also features methods for identifying compoundsthat alter the activity of a nematode fatty acid desaturase-likepolypeptide. The method includes contacting the test compound to thenematode fatty acid desaturase-like polypeptide and detecting a fattyacid desaturase-like activity. A decrease in the level of fatty aciddesaturase-like activity of the polypeptide relative to the level offatty acid desaturase-like activity of the polypeptide in the absence ofthe test compound is an indication that the test compound is aninhibitor of the fatty acid desaturase-like activity. In still anotherembodiment, the method further includes contacting a nematode fatty aciddesaturase polypeptide with a test compound such as an allostericinhibitor, a substrate analog, other analogs, or other types ofinhibitors that prevent binding of the fatty acid desaturase-likepolypeptide to other molecules or proteins (“fatty acid desaturase-likepolypeptide binding partners”). A change in activity or fatty aciddesaturase-like polypeptide binding of proteins normally bound by thefatty acid desaturase is an indication that the test compound is aninhibitor of the fatty acid desaturase-like activity or is an inhibitorof the interaction of the fatty acid desaturase-like polypeptide withone of its binding partners. Such inhibitory compounds are potentiallyselective agents for reducing the viability, growth, development orreproduction of a nematode expressing a fatty acid desaturase-likepolypeptide, e.g., M. incognita, H. glycines, D. immitis, S. stercoralisor R. axei. These methods can also include contacting the test compoundwith a plant or mammalian (e.g., as human) fatty acid desaturase-likepolypeptide; and detecting a fatty acid desaturase-like activity of theplant or mammalian fatty acid desaturase-like polypeptide. A compoundthat decreases nematode fatty acid desaturase activity to a greaterextent than it decreases a plant or mammalian fatty acid desaturase-likepolypeptide activity is a candidate selective inhibitor of nematodeviability, growth or reproduction. A desirable compound can exhibit2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater selectiveactivity against the nematode polypeptide. Any suitable assay can beused to measure fatty acid desaturase activity, including that of Banaset al. (Physiology, Biochemistry and Molecular Biology of Plant Lipids,Williams et al., eds., Kluwer Academic, Dordrecht, Netherlands, 1996,pages 57-59).

[0038] Another featured method is a method of screening for a compoundthat alters an activity of a fatty acid desaturase-like polypeptide oralters binding or regulation of other polypeptides by fatty aciddesaturase. Thus, the activity of the fatty acid desaturase can bemeasured directly by monitoring the substrate or product of the fattyacid desaturase or indirectly by measuring the activity (e.g.,monitoring the substrate or product) of an enzyme that acts downstreamof fatty acid desaturase. Thus, the methods of the invention includeproviding a fatty acid desaturase polypeptide; contacting a testcompound to the polypeptide; and detecting a fatty acid desaturase-likeactivity of the polypeptide or the activity of polypeptides bound orregulated by the fatty acid desaturase, wherein a change in activity ofthe fatty acid desaturase-like polypeptide or other downstreampolypeptides relative to the fatty acid desaturase-like activity of thepolypeptide or downstream polypeptides in the absence of the testcompound is an indication that the test compound alters the activity ofthe polypeptide(s). Similarly, the method includes providing thepolypeptide; contacting a test compound to the polypeptide; anddetecting a fatty acid desaturase-like activity or the resultingunsaturated fatty acid products, wherein a change in activity of fattyacid desaturase-like polypeptides or the resulting unsaturated fattyacid products relative to the fatty acid desaturase-like activity of thepolypeptide or the level of fatty acid products in the absence of thetest compound is an indication that the test compound alters theactivity of the polypeptide(s).

[0039] The methods of the invention can include contacting the testcompound to a plant or mammalian (e.g., a human) fatty aciddesaturase-like polypeptide and measuring the fatty acid desaturase-likeactivity of the plant or mammalian fatty acid desaturase-likepolypeptide or other polypeptides affected or regulated by the fattyacid desaturase or the resulting unsaturated fatty acid products. A testcompound that alters the activity of the nematode fatty aciddesaturase-like polypeptide (or the level of fatty acid products) at agiven concentration and that does not substantially alter the activityof the plant or mammalian fatty acid desaturase-like polypeptide,downstream polypeptides, or level of fatty acid products at the samegiven concentration can be identified. Thus, the methods of theinvention can be used to identify candidate compounds that arerelatively selective for one or more nematode fatty acid desaturase-likepolypeptides relative to one or more mammalian and or plant fatty aciddesaturase-like polypeptides. An additional method includes screeningfor both binding to a fatty acid desaturase-like polypeptide and for analteration in the activity of a fatty acid desaturase-like polypeptide.

[0040] The methods of the invention include the identification ofcompounds that inhibit both nematode and plant fatty aciddesaturase-like polypeptides. Such compounds can be useful for treatmentof prevention of nematode infection of plants because plants are oftennot significantly impaired by inhibition of the activity of a fatty aciddesaturase-like polypeptide. Moreover, such inhibitors can beadministered to a mammal for treatment or prevention of infection by anematode.

[0041] Yet another featured method is a method of screening for acompound that alters the viability or fitness of a transgenic cell ororganism (e.g., a nematode). The transgenic cell or organism has atransgene that expresses a fatty acid desaturase-like polypeptide. Themethod includes contacting a test compound to the transgenic cell ororganism and detecting changes in the viability or fitness of thetransgenic cell or organism. This alteration in viability or fitness canbe measured relative to an otherwise identical cell or organism thatdoes not harbor the transgene.

[0042] The invention also features compounds that are relativelyselective inhibitors of one or more nematode fatty acid desaturase-likepolypeptides relative to one or more plant or animal fatty aciddesaturase-like polypeptides. The compounds can have a K_(i) for anematode fatty acid desaturase that is 10-fold, 100-fold, 1,000-fold ormore lower than for a plant or animal fatty acid desaturase-likepolypeptides, e.g., a host plant or host animal of the nematode. Theinvention further features relatively non-selective inhibitors as wellas completely non-selective inhibitors.

[0043] Also featured is a method of screening for a compound that altersthe expression of a nematode nucleic acid encoding a fatty aciddesaturase-like polypeptide or nucleic acid encoding a nematode fattyacid desaturase-like polypeptide, e.g., a nucleic acid encoding a M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like polypeptide. The method includes contacting a cell,e.g., a nematode cell, with a test compound and detecting expression ofa nematode nucleic acid encoding a fatty acid desaturase-likepolypeptide, e.g., by hybridization to a probe complementary to thenematode mRNA encoding an fatty acid desaturase-like polypeptide or bycontacting polypeptides isolated from the cell with a compound, e.g.,antibody that binds a fatty acid desaturase-like polypeptide.

[0044] In yet another aspect, the invention features a method oftreating a disorder (e.g., an infection) caused by a nematode, e.g., M.incognita, H. glycines, D. immitis or S. stercoralis, in a subject,e.g., a host plant or host animal. The method includes administering tothe subject an effective amount of an inhibitor of a fatty aciddesaturase-like polypeptide activity or an inhibitor of expression of afatty acid desaturase-like polypeptide. Non-limiting examples of suchinhibitors include: an antisense nucleic acid (or PNA) to a fatty aciddesaturase-like nucleic acid, an antibody to a fatty aciddesaturase-like polypeptide, an analog of a natural substrate of a fattyacid desaturase, a fatty acid, or a small molecule identified as a fattyacid desaturase-like polypeptide inhibitor, e.g., an inhibitoridentified by a method described herein.

[0045] In another aspect, methods for desaturating fatty acids to Δ2fatty acids are provided. Such methods can include the steps of: (a)providing a cell that harbors a fatty acid desaturase polypeptide; and(b) growing the cell under conditions in which the fatty acid desaturasepolypeptide desaturates a fatty acid to produce a corresponding Δ2unsaturated fatty acid.

[0046] In still another aspect, methods of inhibiting nematode (e.g., M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei) Δ12 fattyacid desaturase(s) are provided. Such methods can include the steps of:(a) providing a nematode that expresses a Δ12 fatty acid desaturase-likeenzyme; (b) contacting the nematode with fatty acid analogs or othercompounds that inhibit the enzyme. Also provided are methods of rescuingthe effect of the inhibitor. Such methods comprise the steps of: (a)inhibiting the enzyme and (b) providing Δ12 unsaturated fatty acidsexogenously.

[0047] In another aspect, methods of reducing the viability or fecundityor slowing the growth or development or inhibiting the infectivity of anematode using a fatty acid analog or inhibitor of a fatty aciddesaturase are provided. Such methods comprise the steps of (a)providing a nematode that expresses a Δ12 fatty acid desaturase; (b)contacting the nematode with specific inhibitory fatty acid analogs orinhibitors of a Δ12 fatty acid desaturase; (c) reducing the viability orfecundity of the nematode. Also provided are methods of rescuing theeffect of the fatty acid desaturase inhibitors or other inhibitors. Suchmethods can involve contacting the nematode with Δ12 unsaturated fattyacids exogenously.

[0048] In another aspect, methods of inhibiting a Δ12 fatty aciddesaturase using RNA interference methods are provided. Such methodscomprise the steps of (a) providing a nematode that contains a Δ12 fattyacid desaturase like gene; (b) contacting the nematode with doublestranded RNA (dsRNA). Such methods can be used to reduce viability orfecundity, to slow growth or development, or to inhibit infectivity. Inanother aspect, methods of rescuing the effect of RNA interference bysupplying specific Δ12 unsaturated fatty acids are provided.

[0049] The methods of the invention include a method for identifying aninhibitor of a fatty acid desaturase-like polypeptide, the methodcomprising: (a) providing a cell expressing a fatty acid desaturase-likepolypeptide; (b) contacting the cell with a test compound; (c) measuringthe fatty acid desaturase-like polypeptide activity of the cell, whereina change in fatty acid desaturase-like polypeptide activity of the cellrelative to the fatty acid desaturase-like polypeptide activity of thecell in the absence of the test compound is an indication that the testcompound alters the activity of the fatty acid desaturase-likepolypeptide.

[0050] The methods of the invention further include a method foridentifying an inhibitor of a fatty acid desaturase-like polypeptide,the method comprising: (a) providing a nematode expressing a fatty aciddesaturase-like polypeptide; (b) contacting the nematode with a testcompound; (c) measuring the fatty acid desaturase-like polypeptideactivity of the nematode, wherein a change in fatty acid desaturase-likepolypeptide activity of the nematode relative to the fatty aciddesaturase-like polypeptide activity of the nematode in the absence ofthe test compound is an indication that the test compound alters theactivity of the fatty acid desaturase-like polypeptide.

[0051] Another method for identifying an inhibitor of a fatty aciddesaturase-like polypeptide comprises: (a) providing a cell expressing afatty acid desaturase-like polypeptide; (b) contacting the cell with atest compound; (c) measuring the viability of the cell in the presenceof the test compound; and (d) comparing the viability of the cell in thepresence of the test compound to the viability of the cell in thepresence of the test compound and a product of the fatty aciddesaturase-like polypeptide, wherein greater viability in the presenceof the test compound and the product compared to viability in thepresence of the test compound is an indication that the test compoundalters the activity of the fatty acid desaturase-like polypeptide. Theinvention features a method for identifying an inhibitor of a fatty aciddesaturase-like polypeptide, the method comprising: (a) providing anematode expressing a fatty acid desaturase-like polypeptide; (b)contacting the nematode with a test compound; (c) measuring theviability or the fecundity of the nematode in the presence of the testcompound; and (d) comparing the viability or fecundity of the nematodein the presence of the test compound to the viability or fecundity ofthe nematode in the presence of the test compound and a product of thefatty acid desaturase-like polypeptide, wherein greater viability orfecundity in the presence of the test compound and the product comparedto viability or fecundity in the presence of the test compound is anindication that the test compound alters the activity of the fatty aciddesaturase-like polypeptide. In preferred embodiments the product islinoleic acid.

[0052] The invention also features inhibitors identified by thescreening methods disclosed herein.

[0053] The invention features a method for reducing the viability,growth, or fecundity of a nematode, the method comprising exposing thenematode to an agent that inhibits the activity of a fatty aciddesaturase-like polypeptide (e.g., a Δ12 fatty acid desaturase) and amethod for protecting a plant from a nematode infection, the methodcomprising applying to the plant or to seeds of the plant an inhibitorof a nematode fatty acid desaturase-like polypeptide. The invention alsofeatures a method for protecting a mammal from a nematode infection, themethod comprising administering to the mammal an inhibitor of a nematodefatty acid desaturase-like polypeptide (e.g., a Δ12 fatty aciddesaturase). In preferred embodiments the inhibitor does notsignificantly inhibit the activity of a fatty acid desaturase-likepolypeptide expressed by the plant or at least does not do so to theextent that the growth of the plant is impaired.

[0054] A “purified polypeptide”, as used herein, refers to a polypeptidethat has been separated from other proteins, lipids, and nucleic acidswith which it is naturally associated. The polypeptide can constitute atleast 10, 20, 50 70, 80 or 95% by dry weight of the purifiedpreparation.

[0055] An “isolated nucleic acid” is a nucleic acid, the structure ofwhich is not identical to that of any naturally occurring nucleic acid,or to that of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample: (a) a DNA which is part of a naturally occurring genomic DNAmolecule but is not flanked by both of the nucleic acid sequences thatflank that part of the molecule in the genome of the organism in whichit naturally occurs; (b) a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones in a DNA library such as a cDNA or genomic DNAlibrary. Isolated nucleic acid molecules according to the presentinvention further include molecules produced synthetically, as well asany nucleic acids that have been altered chemically and/or that havemodified backbones.

[0056] Although the phrase “nucleic acid molecule” primarily refers tothe physical nucleic acid molecule and the phrase “nucleic acidsequence” refers to the sequence of the nucleotides in the nucleic acidmolecule, the two phrases can be used interchangeably.

[0057] The term “substantially pure” as used herein in reference to agiven polypeptide means that the polypeptide is substantially free fromother biological macromolecules. The substantially pure polypeptide isat least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight.Purity can be measured by any appropriate standard method, for example,by column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

[0058] The “percent identity” of two amino acid sequences or of twonucleic acids is determined using the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLASTN and BLASTX programs (version 2.0 and2.1) of Altschul et al. (1990). J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the BLASTX program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Wheregaps exist between two sequences, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs for the determination ofpercent identity of amino acid sequences or nucleotide sequences, thedefault parameters of the respective programs can be used. The programsare available on the Internet at: www.ncbi.nlm.nih.gov.

[0059] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one or more subject polypeptides), which ispartly or entirely heterologous, i.e., foreign, to the transgenic plant,animal, or cell into which it is introduced, or, is homologous to anendogenous gene of the transgenic plant, animal, or cell into which itis introduced, but which is designed to be inserted, or is inserted,into the plant's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can include one or more transcriptionalregulatory sequences and other nucleic acid sequences, such as introns,that may be necessary for optimal expression of the selected nucleicacid, all operably linked to the selected nucleic acid, and may includean enhancer sequence.

[0060] As used herein, the term “transgenic cell” refers to a cellcontaining a transgene.

[0061] As used herein, a “transgenic plant” is any plant in which one ormore, or all, of the cells of the plant includes a transgene. Thetransgene can be introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by T-DNA mediated transfer, electroporation, orprotoplast transformation. The transgene may be integrated within achromosome, or it may be extrachromosomally replicating DNA.

[0062] As used herein, the term “tissue-specific promoter” means a DNAsequence that serves as a promoter, i.e., regulates expression of aselected DNA sequence operably linked to the promoter, and which effectsexpression of the selected DNA sequence in specific cells of a tissue,such as a leaf, root, seed, or stem.

[0063] As used herein, the terms “hybridizes under stringent conditions”and “hybridizes under high stringency conditions” refer to conditionsfor hybridization in 6× sodium chloride/sodium citrate (SSC) buffer atabout 45° C., followed by two washes in 0.2× SSC buffer, 0.1% SDS at 60°C. or 65° C. As used herein, the term “hybridizes under low stringencyconditions” refers to conditions for hybridization in 6× SSC buffer atabout 45° C., followed by two washes in 6× SSC buffer, 0.1% (w/v) SDS at50° C.

[0064] A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence. As used herein, an agent with “anthelminticactivity” is an agent, which when tested, has measurablenematode-killing activity or results in reduced fertility or sterilityin the nematodes such that fewer viable or no offspring result, orcompromises the ability of the nematode to infect or reproduce in itshost, or interferes with the growth or development of a nematode. In theassay, the agent is combined with nematodes, e.g., in a well ofmicrotiter dish having agar media or in the soil containing the agent.Staged adult nematodes are placed on the media. The time of survival,viability of offspring, and/or the movement of the nematodes aremeasured. An agent with “anthelminthic activity” can, for example,reduce the survival time of adult nematodes relative to unexposedsimilarly staged adults, e.g., by about 20%, 40%, 60%, 80%, or more. Inthe alternative, an agent with “anthelminthic activity” may also causethe nematodes to cease replicating, regenerating, and/or producingviable progeny, e.g., by about 20%, 40%, 60%, 80%, or more.

[0065] As used herein, the term “binding” refers to the ability of afirst compound and a second compound that are not covalently linked tophysically interact. The apparent dissociation constant for a bindingevent can be 1 mM or less, for example, 10 nM, 1 nM, 0.1 nM or less.

[0066] As used herein, the term “binds specifically” refers to theability of an antibody to discriminate between a target ligand and anon-target ligand such that the antibody binds to the target ligand andnot to the non-target ligand when simultaneously exposed to both thegiven ligand and non-target ligand, and when the target ligand and thenon-target ligand are both present in molar excess over the antibody.

[0067] As used herein, the term “altering an activity” refers to achange in level, either an increase or a decrease in the activity,(e.g., an increase or decrease in the ability of the polypeptide to bindor regulate other polypeptides or molecules) particularly a fatty aciddesaturase-like or fatty acid desaturase activity (e.g., the ability tointroduce a double bond at the Δ12 position of a fatty acid). The changecan be detected in a qualitative or quantitative observation. If aquantitative observation is made, and if a comprehensive analysis isperformed over a plurality of observations, one skilled in the art canapply routine statistical analysis to identify modulations where a levelis changed and where the statistical parameter, the p value, is, forexample, less than 0.05.

[0068] In part, the nematode fatty acid desaturase proteins and nucleicacids described herein are novel targets for anti-nematode vaccines,pesticides, and drugs. Inhibition of these molecules can provide meansof inhibiting nematode metabolism, growth, viability, fecundity,development, infectivity and/or the nematode life-cycle.

[0069] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0070]FIG. 1 depicts the cDNA sequence of a M. incognita fatty aciddesaturase (SEQ ID NO: 1), its corresponding encoded amino acid sequence(SEQ ID NO: 8).

[0071]FIG. 2 depicts the cDNA sequence of a second M. incognita fattyacid desaturase (SEQ ID NO: 2), its corresponding encoded amino acidsequence (SEQ ID NO: 9).

[0072]FIG. 3 depicts the cDNA sequence of a third M. incognita fattyacid desaturase (SEQ ID NO: 3), its corresponding encoded amino acidsequence (SEQ ID NO: 10).

[0073]FIG. 4 depicts the cDNA sequence of a H. glycines fatty aciddesaturase (SEQ ID NO: 4), its corresponding encoded amino acid sequence(SEQ ID NO: 11).

[0074]FIG. 5 depicts the partial cDNA sequence of a D. immitis fattyacid desaturase (SEQ ID NO: 5), its corresponding encoded amino acidsequence (SEQ ID NO: 12).

[0075]FIG. 6 depicts the cDNA sequence of a S. stercoralis fatty aciddesaturase (SEQ ID NO: 6), its corresponding encoded amino acid sequence(SEQ ID NO: 13).

[0076]FIG. 7 depicts the cDNA sequence of a R. axei fatty aciddesaturase (SEQ ID NO: 7), its corresponding encoded amino acid sequence(SEQ ID NO: 14).

[0077]FIG. 8 depicts an alignment of the sequences of M. incognita, H.glycines, D. immitis, S. stercoralis and R. axei fatty aciddesaturase-like polypeptides (SEQ ID NO: 8, 9, 10, 11, 12, 13 and 14 anda C. elegans Δ12 fatty acid desaturase polypeptide (SEQ ID NO: 32 (lowersequence)).

[0078]FIG. 9 is a photograph of C. elegans grown on oleic acid.

[0079]FIG. 10 is a photograph of C. elegans grown on linoleic acid.

[0080]FIG. 11 is a photograph of C. elegans grown on ricinoleic acid.

[0081]FIG. 12 is a photograph of C. elegans grown on vernolic acid.

DETAILED DESCRIPTION

[0082] Described below is the identification of M. incognita, H.glycines, D. immitis, S. stercoralis and R. axei fatty acid desaturasecDNAs (SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7) and the polypeptides theyencode (SEQ ID NO: 8, 9, 10, 11, 12, 13 and 14). The fatty aciddesaturases are Δ12 fatty acid desaturases. Also described below areexperiments demonstrating that the fatty acid desaturase is essentialfor nematode viability. Also described below are inhibitors of the fattyacid desaturase. Certain sequence information for the fatty aciddesaturase genes described herein is summarized in Table 1, below. TABLE1 cDNAs Identified Species cDNA Polypeptide Figure M. incognita SEQ IDNO: 1 SEQ ID NO: 8 M. incognita SEQ ID NO: 2 SEQ ID NO: 9 M. incognitaSEQ ID NO: 3 SEQ ID NO: 10 H. glycines SEQ ID NO: 4 SEQ ID NO: 11 D.immitis SEQ ID NO: 5 SEQ ID NO: 12 S. stercoralis SEQ ID NO: 6 SEQ IDNO: 13 R. axei SEQ ID NO: 7 SEQ ID NO: 14

[0083] Unsaturated fatty acids are essential to the proper functioningof biological membranes. At physiological temperatures, polarglycerolipids that contain only saturated fatty acids cannot form theliquid-crystalline bilayer that is the fundamental structure ofbiological membranes. The introduction of an appropriate number ofdouble bonds (a process referred to as desaturation) into the fattyacids of membrane glycerolipids decreases the temperature of thetransition from the gel to the liquid-crystalline phase and providesmembranes with necessary fluidity. Fluidity of the membrane is importantfor maintaining the barrier properties of the lipid bilayer and for theactivation and function of certain membrane bound enzymes. There is alsoevidence that unsaturation confers some protection to ethanol andoxidative stress, suggesting that the degree of unsaturation of membranefatty acids has importance beyond temperature adaptation. Unsaturatedfatty acids are also precursors of polyunsaturated acids (PUFAs)arachidonic and eicosapentaenoic acids in animals, which are importantsources of prostaglandins. These molecules are local hormones that alterthe activities of the cells in which they are synthesized and inadjoining cells, mediating processes in reproduction, immunity,neurophysiology, thermobiology, and ion and fluid transport.

[0084] The ability of cells to modulate the degree of unsaturation intheir membranes is primarily determined by the action of fatty aciddesaturases. Desaturase enzymes introduce unsaturated bonds at specificpositions in their fatty acyl chain substrates, using molecular oxygenand reducing equivalents from NADH (or NADPH) to catalyze the insertionof double bonds. In many systems, the reaction uses a short electrontransport chain consisting of NAD(P)H, cytochrome b5 reductase, andcytochrome b5, to shuttle electrons from NAD(P)H and the carbon-carbonsingle bond to oxygen, forming water and a double bond (C═C). Manyeukaryotic desaturases are endoplasmic reticulum (ER) bound non-hemediiron-oxo proteins which contain three conserved histidine-rich motifsand two long stretches of hydrophobic residues. These hydrophobic alphahelical domains are thought to position the protein with its bulkexposed to the cytosolic face of the ER and to organize the active sitehistidines to appropriately coordinate the active diiron-oxo moiety.

[0085] While most eukaryotic organisms, including mammals, can introducea double bond into an 18-carbon fatty acid at the Δ9 position, mammalsare incapable of inserting double bonds at the Δ12 or Δ15 positions. Forthis reason, linoleate (18:2 Δ9,12) and linolenate (18:3 Δ9,12,15) mustbe obtained from the diet and, thus, are termed essential fatty acids.These dietary fatty acids come predominately from plant sources, sinceflowering plants readily desaturate the Δ12 and the Δ15 positions.Certain animals, including some insects and nematodes, can synthesize denovo all their component fatty acids including linoleate and linolenate(Watts and Browse (2002) Proc Natl Acad Sci USA, 99(9):5854-9; Borgesonet al. (1990) Biochim Biophys Acta. 1047(2):135-40; Cripps et al. (1990)Arch Biochem Biophys. 278(1):46-51). The nematode C. elegans, forexample, can synthesize de novo a broad range of polyunsaturated fattyacids including arachidonic acid and eicosapentaenoic acids, a featurenot shared by either mammals or flowering plants (Tanaka et al. (1999)Eur J. Biochem. 263(1):189-95).

[0086] The C. elegans desaturase genefat-2 has been expressed in S.cerevisiae and shown to be a Δ12 fatty acid desaturase. This enzymeintroduces a double bond between the 12^(th) and the 13^(th) carbons(from the carboxylate end) and can convert the mono-unsaturated oleate(18:1 Δ9) and palmitoleate (16:1Δ9) to the di-unsaturated linoleate(18:2Δ9,12) and 16:2 Δ9,12 fatty acids, respectively.

[0087] The nematode Δ12 enzymes are potentially good targets foranti-nematode compounds for several reasons. Firstly, the enzymes appearto be phylogenetically diverged from their homologs in plants, havingless than 40% pairwise sequence identity at the amino acid level andphylogenetic analyses demonstrate clustering of nematode Δ12 and ω-3desaturases away from homologs in plants. Experiments with bothtransgenic Arabidopsis and soybeans reveal that plants can toleratesignificant reductions in Δ12 fatty acid desaturase activity, suggestingthat inhibitors of desaturases would likely not be toxic to plants(Singh et al. (2000) Biochem. Society Trans. 28: 940-942; Lee et al.(1998) Science 280:915-918). In addition, as mentioned above, mammalsare thought not to have Δ12 fatty acid desaturases. Thus, inhibitors ofthe enzyme are likely to be non-toxic to mammals. Importantly, asdetailed herein, a fatty acid desaturase of nematodes has been shown tobe essential to their viability, both through inhibitor and RNA-mediatedinterference studies. Thus, Δ12 fatty acid desaturases could serve asideal targets for anti-nematode control, as inhibitors of the enzymecould specifically target nematodes while leaving their animal and planthosts unharmed.

[0088] Numerous analogs of fatty acids exist and some may act asspecific inhibitors of enzymes such as desaturases that act on fattyacids, a fact that could be exploited for development of anti-nematodecompounds. Sterculic acid, a cyclopropenoid fatty acid analog of oleicacid, is a potent inhibitor of Δ9 fatty acid desaturases (Schmid &Patterson (1998) Lipids 23(3):248-52; Waltermann & Steinbuchel (2000)FEMS Microbiol Lett. 190(1):45-50). It has also been speculated thatcyclopropenoid analogs of linoleic acid may similarly inhibit Δ12 fattyacid desaturases (Dulayymi et al. (1997) Tetrahedron 53(3):1099-1110).It is worth noting however that malvalate, a Δ8 cyclopropene fatty acid,seems to be equally inhibitory to Δ9 desaturases in some systems,(Schmid & Patterson (1998) Lipids 23(3):248-52), demonstrating howdifficult it is to predict inhibitory profiles for some fatty acidanalogs. Thia fatty acid analogs (i.e., sulfur containing fatty acids)are also potential inhibitors of fatty acid desaturases (Skrede et al.(1997) Biochim Biophys Acta 1344(2):115-131; Hovik et al. (1997) BiochimBiophys Acta 1349(3):251-256) as are trans fatty acids (Choi et al.(2001) Biochem Biophys Res Commun 284(3):689-93). However, thespecificity and pesticidal activity of these analogs is again difficultto predict (Beach et al. (1989) Mol Biochem Parasitol 35(1):57-66).

[0089] Certain fatty acids are also specific receptor antagonists(Yagaloff (1995) Prostaglandins Leukot Essent Fatty Acids 52(5):293-7).

[0090] Other analogs of linoleic acid that may also be specific Δ12inhibitors include the epoxy fatty acid (vernolic acid), the acetylenicfatty acid (crepenynic acid), 12-oxo-9(Z)-octadecenoic acid methyl esteror the hydroxy fatty acids (ricinoleic and ricinelaidic acid).Inhibitors that interfere with Δ12 fatty acid desaturase activity areexpected to be toxic to nematodes. Importantly, fatty acid analogs suchas ricinoleic, ricinelaidic, vernolic and crepenynic acid methyl estersdo not appear to be toxic (or are very much less toxic) to at least someplants and are predicted not to be toxic (or are very much less toxic)to at least some animals, including mammals. Such fatty acid analogscould potentially be used in the development of nematode control agents.

[0091] Although previously expressed in plants, fatty acid analogs suchas crepenynate, ricinoleate and vernolate acids were not thought to bespecific inhibitors of the endogenous plant Δ12 desaturase (Broun &Somerville (1997) Plant. Physiol. 113:933-942; Singh et al. (2000)Biochem. Society Trans. 28(6): 940-942). Changes in the ratio of oleateto linoleate in plants expressing the genes for these analogs wasinstead attributed to a negative interaction between the enzymesinvolved (Singh et al. (2001) Planta 212: 872-879). Addition ofricinoleate exogenously to Neurospora crassa results in a significantdecrease in oleate (C18:1) and an increase in linolenate (C18:3) againproviding no indication that compounds like ricinoleate were in factspecific Δ12 desaturase inhibitors (Goodrich-Tanrikulu et al. (1996)Appl Microbiol Biotechnol. 46(4):382-7).

[0092] We made the surprising discovery that methyl esters of certainfatty acid analogs (e.g., ricinoleate, vernolate) are nematicidal andhave activity consistent with that of specific inhibitors of nematodeΔ12 desaturases. The fatty acid methyl esters show significantlyenhanced activity over other eighteen carbon fatty acid esters such asoleate, elaidate and linoleate. In contrast to short chain seeminglynon-specific pesticidal fatty acid esters such as laurate andpelargonate, the fatty acid analogs that are predicted Δ12 desaturaseinhibitors show dramatically reduced phytoxicity and can therefore beused effectively while minimizing undesirable damage to non-targetorganisms.

[0093] Fatty acid-based analogs or other types of inhibitors may besupplied to plants exogenously, through sprays for example. It is alsopossible to provide inhibitors through a host organism or an organism onwhich the nematode feeds. For example, a host cell that does notnaturally produce an inhibitor of the M. incognita, H. glycines, D.immitis and S. stercoralis fatty acid desaturase-like polypeptides canbe transformed with enzymes capable of making inhibitory analogs andprovided with appropriate precursor chemicals exogenously.Alternatively, the active inhibitors and precursors can be madeendogenously by the expression of the appropriate enzymes. In addition,yeast or other organisms can be modified to produce inhibitors.Nematodes that feed on such organisms would then be exposed to theinhibitors.

[0094] In one embodiment, transgenic cells and/or organisms could begenerated that produce enzymes active on fatty acids (e.g.,desaturating, hydroxylating, conjugating, and/or epoxygenating enzymes).Such enzymes may be expressed, for example, in plants, vertebrates,and/or nematodes. These enzymes may produce fatty acids, analogs, orother inhibitors that can then act as specific inhibitors for otherenzymes such as a fatty acid desaturase (e.g., a Δ12 epoxygenase fromCrepis palaestina produces vernolic acid, a Δ12 desaturase inhibitor, intransgenic Arabidopsis) (Singh et. al. (2000) Biochem. Society Trans.28:940-942; Lee et al. (1998) Science 280:915-918).

[0095] More generally, a recombinant expression vector capable ofexpressing an enzyme active on fatty acids could be transformed into ahost cell of an organism that is parasitized by a parasitic nematode,(M. incognita, H. glycines, D. immitis or S. stercoralis, for example).Fatty acid analogs that act as inhibitors of M. incognita, H. glycines,D. immitis or S. stercoralis Δ12 fatty acid desaturases, for example,can then be produced in the host cell or organism. In this manner, Δ12fatty acid desaturases from feeding parasitic nematodes (e.g., M.incognita, H. glycines, D. immitis or S. stercoralis) could be renderedinactive by the fatty acid analog.

[0096] In another embodiment, a recombinant expression vector harboringa Δ12 fatty acid desaturase-like polypeptide from, for example, M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei, can beused to produce a recombinant fatty acid desaturase polypeptide that isfunctional in a cell, plant or animal, and that can desaturate fattyacids that are normally produced by the cell, plant or animal or thatare provided exogenously to the cell, plant or animal to thecorresponding Δ12 fatty acid. In this way, a cell, plant or animal canbe produced that has a higher proportion of Δ12 unsaturated fatty acidsthan an otherwise similar cell, plant, or animal lacking the recombinantfatty acid desaturase polypeptide. In this way, a cell, plant or animalthat has increased resistance to a Δ12 fatty acid desaturase inhibitorcan be produced.

[0097] A recombinant Δ12 fatty acid desaturase, e.g., a M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase,may also be useful for producing lipids having a higher proportion ofΔ12 unsaturated fatty acids, whether by means of recombinant expressionin a cell or in an industrial process using purified nematode Δ12 fattyacid desaturase polypeptide. Such lipids are useful as food oils, asnutritional supplements, and as chemical feedstocks, for example.

[0098] The following examples are therefore to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. All of the publications cited herein are herebyincorporated by reference in their entirety.

EXAMPLES

[0099] TBLASTN searches of a database of nematode EST sequences(McCarter et al. (1999) Washington University Nematode EST Project) withC. elegans Δ12 fatty acid desaturase (FAT-2) gene sequence (GenBank®Accession No: AAF63745: ID No: 7546993) identified two EST's (AW783527and AW871151) that are predicted to encode at least a portion of a Δ12Fatty Acid Desaturase-like enzymes (Δ12 FAT) in one nematode species, M.incognita.

[0100] Full Length Δ12 Fatty Acid Desaturase-Like cDNA Sequences

[0101] The plasmid clone corresponding to the M. incognita EST sequenceAW783527 was obtained from the Genome Sequencing Center (St. Louis,Mo.). This plasmid clone was designated Div249. The cDNA insert in theplasmid was sequenced in its entirety. Unless otherwise indicated, allnucleotide sequences determined herein were sequenced with an automatedDNA sequencer (such as model 373 from Applied Biosystems, Inc.) usingprocesses well known to those skilled in the art. Primers used forsequencing are listed in Table 1 (see below). Partial sequence data forthe M. incognita Δ12 FAT was obtained from Div249, including nucleotidesequence for codons 90-397 and additional 3′ untranslated sequence. Theclone lacked the first 89 codons of the M. incognita Δ12 FAT, as well asthe 5′ untranslated region.

[0102] The following methods were used to obtain the full-lengthnematode Δ12 FAT gene and to determine its complete sequence. First, RNAwas obtained from plant parasitic nematodes, which are maintained ongreenhouse pot cultures depending on nematode preference. Root KnotNematodes (Meloidogyne sp) were propagated on Rutgers tomato (Burpee).Total RNA was isolated using the TRIZOL reagent (Gibco BRL). Briefly, 2ml of packed worms were combined with 8 ml TRIZOL reagent andsolubilized by vortexing. Following 5 minutes of incubation at roomtemperature, the samples are divided into smaller volumes and spun at14,000×g for 10 minutes at 4° C. to remove insoluble material. Theliquid phase was extracted with 200 μl of chloroform, and the upperaqueous phase was removed to a fresh tube. The RNA was precipitated bythe addition of 500 μl of isopropanol and centrifuged to pellet. Theaqueous phase was carefully removed, and the pellet was washed in 75%ethanol and spun to re-collect the RNA pellet. The supernatant wascarefully removed, and the pellet was air dried for 10 minutes. The RNApellet was resuspended in 50 μl of DEPC-H₂O and analyzed byspectrophotometry at 260 and 280 nm to determine yield and purity.Yields could be 1-4 mg of total RNA from 2 ml of packed worms.

[0103] To obtain the missing 5′ sequence of the M. incognita Δ12 FATgene, the 5′ RACE technique was applied, and SL1 PCR was performed usingfirst strand cDNA from M. incognita as a template. Briefly, SL1 PCRutilizes the observation, that unlike most eukaryotic mRNAs, manynematode mRNA molecules contain a common leader sequence (5′ ggg ttt aattac cca agt ttg a 3′; SEQ ID NO: 17) transpliced to their 5′ ends. Ifthis sequence is present on the 5′ end of a cDNA, that cDNA can beamplified using PCR with a primer that binds to the SL1 transplicedleader and a gene-specific primer near the 3′ end of the cDNA.

[0104] Briefly, following the instructions provided by Life TechnologiescDNA synthesis kit, first strand cDNA synthesis was performed on totalnematode RNA using SuperScript™ II Reverse Transcriptase and an oligo-dTprimer (which anneals to the natural poly A tail found on the 3′ end ofall eukaryotic mRNA). RNase H was then used to degrade the original mRNAtemplate. Following degradation of the original mRNA template, the firststrand cDNA was directly PCR amplified without further purificationusing Taq DNA polymerase, a gene specific primer (FAT10, SEQ ID NO: 19)designed from known sequence that anneals to a site located within thefirst strand cDNA molecule, and the SL1 primer, which is homologous the5′ end of the cDNA of interest. Amplified PCR products were then clonedinto a suitable vector for DNA sequence analysis. This procedure wasperformed to obtain clone Div864. This clone contained codons 1-120 inaddition to 5′ untranslated sequences. Taken together, clones Div249 andDiv864 contain sequences comprising the complete open reading frame ofthe Δ12 FAT gene from M. incognita.

[0105] To obtain the complete Δ12 fatty acid desaturase gene from M.incognita on one clone, primers FAT30 and FAT31 were designed to amplifythe complete open reading frame. Following PCR amplification, severalindependent clones were obtained. DNA sequence analysis revealed thattwo very similar but distinct fatty acid desaturase genes had beenidentified. Along with the gene sequence reported for SEQ ID NO: 1 (1191nucleotide ORF, 397 amino acid polypeptide, FIG. 1), we also identifiedSEQ ID NO: 2 (1239 nucleotide ORF, 413 amino acid polypeptide, FIG. 2).The two genes are very similar and are identical in the regionshomologous to primers FAT30 and FAT31. Clones containing sequencesidentical to SEQ ID NO: 1 included Div1456 and Div1459. Clonescontaining sequences identical to SEQ ID NO: 2 included Div1458 andDiv1463. While cloning the first two M. incognita Δ12 fatty aciddesaturase genes, a third Δ12 fatty acid desaturase gene fragment fromM. incognita was obtained by PCR amplification using the FAT10/SL1primer combination, resulting in clone Div866. This clone containscodons 1-108 of the third M. incognita Δ12 fatty acid desaturase gene.In order to obtain the complete gene sequence, a gene-specific primer(FAT23, SEQ ID NO: 23) designed to a known sequence that anneals to asite located within the first strand cDNA molecule, and an oligo dTprimer, which is homologous to the 3′ end of the cDNA of interest wereused. Amplified PCR products were then cloned into a suitable vector forDNA sequence analysis. This procedure was performed to obtain cloneDiv2727. This clone contains codons 96-387 in addition to 3′untranslated sequences. Taken together, clones Div866 and Div2727contain sequences comprising the complete open reading of the third Δ12fatty acid desaturase gene from M. incognita. The gene sequence reportedfor SEQ ID NO: 3 (1161 nucleotide ORF, 387 amino acid polypeptide, FIG.3) is very similar to the first two M. incognita Δ12 fatty aciddesaturase genes. The first two predicted Δ12 fatty acid desaturasepolypeptides (SEQ ID NO: 8 and 9) are approximately 92% identical toeach other and approximately 57% and 56% identical to the C. elegansfatty acid desaturase (SEQ ID NO: 32), respectively, and areapproximately 69% and 70% identical to the third M. incognita predictedΔ12 fatty acid desaturase polypeptide (SEQ ID NO: 10), respectively. Thethird predicted Δ12 FAT polypeptide (SEQ ID NO: 10) is approximately 51%identical to the C. elegans Δ12 fatty acid desaturase (SEQ ID NO: 32)and 69% similar.

[0106] In order to obtain the H. glycines Δ12 fatty acid desaturasegene, the 5′ RACE technique was applied, and SL1 PCR was performed usingfirst strand cDNA from H. glycines as a template (cDNA synthesisexplained above). The first strand cDNA was directly PCR amplified usingthe SL1 primer and a gene specific degenerate primer (FAT33, SEQ ID NO:24) designed to anneal to region of strong homology shared across manynematode desaturase genes. Amplified PCR products were then cloned intoa suitable vector for DNA sequence analysis. This procedure wasperformed to obtain clone Div1870. This clone contained codons 1-193 inaddition to 5′ untranslated sequences. To obtain the 3′ sequence of thegene, the 3′ RACE technique was applied. The first strand cDNA wasdirectly PCR amplified using a gene specific primer (FAT44, SEQ ID NO:25) designed from known sequence that anneals within the first strandcDNA molecule of interest, and an oligo dT primer, which is homologousto the 3′ end of the cDNA of interest. This procedure was performed togenerate clone Div2724, which contains codons 144-389 in addition to 3′untranslated sequences. Taken together, clones Div1870 and Div2724contain sequences comprising the complete open reading frame of the Δ12fatty acid desaturase gene of H. glycines. The predicted Δ12 fatty aciddesaturase polypeptide reported for SEQ ID NO: 11, (1167 nucleotide ORF,389 amino acid polypeptide, FIG. 4) is approximately 56% identical and73% similar to the C. elegans fatty acid desaturase (SEQ ID NO: 32).

[0107] In an attempt to obtain the D. immitis Δ12 fatty acid desaturasegene, first strand cDNA, using D. immitis cDNA as a template (cDNAsynthesis described above), was directly PCR amplified, using agene-specific degenerate primer (FAT35, SEQ ID NO: 26) designed toanneal to a region of strong homology shared across many nematodedesaturase genes, and another gene-specific degenerate primer (FT05, SEQID NO: 27), which was predicted to be homologous to a region near the 5′end of the cDNA of interest. Amplified PCR products were then clonedinto a suitable vector for DNA sequence analysis. This procedure wasperformed to obtain clone Div3228, which contains codons 1-224, whichcorrespond to codons 78-301 of the C. elegans fatty acid desaturase (SEQID NO: 32). To acquire the missing 3′ sequence of D. immitis Δ12 fattyacid desaturase, the 3′ RACE technique was applied using a gene-specificprimer (FAT12, SEQ ID NO: 28) designed to a known sequence that annealsto a site located within the first strand cDNA molecule, and an oligo dTprimer, which is homologous to the 3′ end of the cDNA of interest.Amplified PCR products were then cloned into a suitable DNA vector forsequence analysis. This procedure was performed to obtain the cloneDiv3230, which contained the codons 196-289, which correspond to codons273-376 of the C. elegans fatty acid desaturase (SEQ ID NO: 32), inaddition to 3′ untranslated sequences. Taken together, clones Div3228and Div3230 comprise approximately 80% of the complete D. immitis Δ12FAT open reading frame. The 5′ end sequence of this gene has yet to becompleted. The partial Δ12 fatty acid desaturase polypeptide sequencereported for SEQ ID NO: 12 (867 nucleotide ORF, 289 amino acidpolypeptide, FIG. 5) is 62% identical and 75% similar to the C. elegansfatty acid desaturase (SEQ ID NO: 32).

[0108] Plasmid clone, Div3013, corresponding to the S. stercoralis ESTsequence (GenBank® Identification No: 9830288) was obtained from theGenome Sequencing Center (St. Louis, Mo.). The cDNA insert in theplasmid was sequenced in its entirety. Full sequence data for the S.stercoralis Δ12 fatty acid desaturase was obtained from Div3013,including nucleotide sequence for codons 1-368, (the full open readingframe) and additional 5′ and 3′ untranslated sequences. The predictedgene sequence reported for SEQ ID NO: 6 (1104 nucleotide ORF, 368 aminoacid polypeptide, FIG. 6) is approximately 61% identical and 76% similarto the C. elegans fatty acid desaturase (SEQ ID NO: 32).

[0109] In order to obtain the sequence of the R. axei Δ12 fatty aciddesaturase gene, first strand cDNA from R. axei was directly PCRamplified, using a gene-specific degenerate primer (FAT35, SEQ ID NO:26) designed to anneal to region of strong homology shared across manynematode desaturase genes, and another gene-specific degenerate primer(FAT34, SEQ ID NO: 29) designed to anneal to region of strong homologyshared across many nematode desaturase genes near the 3′ end of the cDNAof interest. Amplified PCR products were then cloned into a suitable DNAvector for sequence analysis. This procedure was performed to obtain theclone Div1843, which contained the codons 185-301 (an internal fragmentmissing both the 5′ and 3′ ends of the gene). In order to obtain themissing 5′ sequence of the R. axei Δ12 fatty acid desaturase gene, the5′ RACE technique was applied and SL1 PCR was performed using firststrand cDNA from R. axei as a template (cDNA synthesis described above).The first strand cDNA was directly PCR amplified using a gene specificprimer (FAT40, SEQ. ID. NO. 30) designed from the known sequence ofclone Div1843, that anneals to a site located within the cDNA ofinterest, and the SL1 primer, which is homologous to the 5′ end of manynematode cDNAs. Amplified PCR products were then cloned into a suitablevector for DNA sequence analysis. This procedure was performed to obtainthe clone Div2026, which contained the codons 1-199, in addition to 5′untranslated sequences. To obtain the missing 3′ sequence of the gene,the 3′ RACE technique was applied using a gene specific primer (FAT42,SEQ ID NO: 31) designed from the known sequence of clone Div 1843, thatanneals to a site located within the cDNA of interest, and an oligo dTprimer, which is homologous to the 3′ end of the cDNA of interest.Amplified PCR products were then cloned into a suitable vector for DNAsequence analysis. This procedure was performed to obtain clone Div2149.This clone contained codons 246-374 in addition to 3′ untranslatedsequences. Taken together, clones Div1843, Div2149, and Div2026 containsequences comprising the complete open reading frame of the Δ12 FAT genefrom R. axei. The predicted Δ12 fatty acid desaturase polypeptide genesequence reported for SEQ ID NO: 7, (1122 nucleotide ORF, 374 amino acidpolypeptide, FIG. 7) is approximately 71% identical and 82% similar toC. elegans fatty acid desaturase (SEQ ID NO: 32). TABLE 2 PrimersEmployed in Cloning SEQ ID Name Sequence NO: Homology to T7Gtaatacgactcactatagggc 15 vector polylinker primer T3Aattaaccctcactaaaggg 16 vector polylinker primer SL1Gggtttaattacccaagtttga 17 nematode transpliced leader Oligo dTgagagagagagagagagagaactagtctcgagtttttttttttttttttt 18 universal primerto poly A tail FAT10 5′ aag ttc cgt gcc cac aat c 3′ 19 Mi Δ12 FAT(codons 115-120)* FAT11 5′ gcc aaa aat gag aac cat cg 3′ 20 Mi Δ12 FAT(codons 206-211)* FAT30 5′ atg tct tat ctt gac aca ac 3′ 21 Mi Δ12 FAT(codons 1-6)* FAT31 5′ cta ttt atc ctt ttt att at 3′ 22 Mi Δ12 FAT(codons 392-397)* FAT23 tctatattcgctgttggacacg 23 Mi Δ12 FAT (codons96-102) FAT33 gtrtadatnggrttcca 24 Ce Δ12 FAT (codons 174-179) FAT44ctggtactgcttgctcggca 25 Hg Δ12 FAT (codons 92-97) FAT35aaraaraartgrtgngcnacrtg 26 Ce Δ12 FAT (codons 295-301)^(#) FT05atgggnatgttyggntc 27 Ce Δ12 FAT (codons 81-85)^(#) FT12gtacaaaccattgatcgag 28 Di Δ12 FAT (codons 196-201) FAT34gayggntctcayttytggccntgg 29 Ce Δ12 FAT (codons 185-192)^(#) FAT40ctctatcttcagtcgttgtg 30 Ra Δ12 FAT (codons 179-202) FAT42ggttatcatcacctatctgc 31 Ra Δ12 FAT (codons 246-251)

[0110] Characterization of M. incognita, H. glycines, D. immitis, S.stercoralis and R. axei Δ12 Fatty Acid Desaturase Genes.

[0111] The similarity between the Δ12 fatty acid desaturase proteinsfrom M. incognita, H. glycines, D. immitis, S. stercoralis and R. axeifrom C. elegans is presented as a multiple alignment generated by theClustalX multiple alignment program as described below (FIG. 8).

[0112] The similarity between M. incognita, H. glycines, D. immitis, S.stercoralis and R. axei Δ12 fatty acid desaturase-like sequences andother sequences was also investigated by comparison to sequencedatabases using BLASTP analysis against nr (a non-redundant proteinsequence database available on the Internet at www.ncbi.nlm.nih.gov) andTBLASTN analysis against dbest (an EST sequence database available onthe Internet at www.ncbi.nlm.nih.gov; top 500 hits; E=1e-4). The “Expect(E) value” is the number of sequences that are predicted to align bychance to the query sequence with a score S or greater given the size ofthe database queried. This analysis was used to determine the potentialnumber of plant and vertebrate homologs for each of the nematode fattyacid desaturase-like polypeptides described above. While M. incognita(SEQ ID NO: 1, 2 and SEQ ID NO:3), H. glycines (SEQ ID NO:4), D. immitis(SEQ ID NO:5), S. stercoralis (SEQ ID NO:6) and R. axei (SEQ ID NO:7)fatty acid desaturase-like cDNA sequences had numerous plant hits, theyhad no vertebrate hits in nr or dbest having sufficient sequencesimilarity to meet the threshold E value of 1e-4 (this E valueapproximately corresponds to a threshold for removing sequences having asequence identity of less than about 25% over approximately 100 aminoacids). Accordingly, the M. incognita, H. glycines, D. immitis, S.stercoralis and R. axei fatty acid desaturase-like enzymes of thisinvention do not appear to share significant sequence similarity withthe more common vertebrate fatty acid desaturase enzymes such as the Δ6fatty acid desaturase of Homo sapiens (a member of the FAD family ofdesaturases, GenBank Accession No. NP_(—)068373), the Δ5 fatty aciddesaturase of Homo sapiens (also a member of the FAD family, GenBankAccession No. NP_(—)037534) or other mammalian fatty acid desaturases.

[0113] On the basis of the lack of similarity to vertebrates, the D.immitis and S. stercoralis fatty acid desaturase-like enzymes (e.g., Δ12fatty acid desaturase) are useful targets of inhibitory compoundsselective for some nematodes over their hosts (e.g., humans, animals).While at least some plants have fatty acid desaturases that are somewhathomologous to those in parasitic nematodes, including M. incognita andH. glycines, other criteria make them promising targets for the controlof plant parasitic nematodes, including the fact that at least in somecases, plants can tolerate significant reductions in fatty aciddesaturase activity while, as demonstrated below, nematodes can not.

[0114] Functional predictions were made using four iterations ofPSI-BLAST with the default parameters on the nr database. PSI-BLASTsearches and multiple alignment construction with CLUSTALX demonstratedthat the C. elegans gene (GenBank® Accession No: AAF63745) was a memberof the fatty acid desaturase family. Reciprocal blast searches andphylogenetic trees confirm that the nucleotide sequences in M.incognita, H. glycines, D. immitis, S. stercoralis and R. axei do encodeorthologs of the C. elegans gene and therefore are also likely fattyacid desaturase proteins with the same activity. Protein localizationswere predicted using the TargetP server available on the Internet at:www.cbs.dtu.dk/services/TargetP. The M. incognita fatty acid desaturase(SEQ ID NO: 8, 9 and SEQ ID NO: 10), H. glycines (SEQ ID NO: 11), D.immitis (SEQ ID NO: 12), S. stercoralis (SEQ ID NO: 13) and R. axei (SEQID NO: 14) polypeptides are likely to be cytoplasmic. In addition, theyare predicted to have four transmembrane regions, consistent with themodel that they are membrane bound.

[0115] RNA Mediated Interference (RNAi)

[0116] A double stranded RNA (dsRNA) molecule can be used to inactivatea Δ12 fatty acid desaturase (Δ12 FAT) gene in a cell by a process knownas RNA mediated-interference (Fire et al. (1998) Nature 391:806-811, andGönczy et al. (2000) Nature 408:331-336). The dsRNA molecule can havethe nucleotide sequence of a Δ12 FAT nucleic acid described herein or afragment thereof. The dsRNA molecule can be delivered to nematodes viadirect injection, or by soaking nematodes in aqueous solution containingconcentrated dsRNA, or by raising bacteriovorous nematodes on E. coligenetically engineered to produce the dsRNA molecule.

[0117] RNAi by injection: To examine the effect of inhibiting Δ12 FATactivity, a Δ12 dsRNA was injected into the nematode, basically asdescribed in Mello et al. (1991) EMBO J. 10:3959-3970. Briefly, aplasmid was constructed that contains a portion of the C. elegans Δ12FAT gene sequence, specifically a fragment 651 nucleotides long,containing the entire first exon and terminating just before theconserved intron splice junction between the first exon and firstintron. This construct encodes approximately the first 217 amino acidsof the C. elegans Δ12 FAT gene. Primers were used to specificallyamplify this sequence as a linear dsDNA. Single-stranded RNAs weretranscribed from these fragments using T7 RNA polymerase and SP6 RNApolymerase (the RNAs correspond to the sense and antisense RNA strands).RNA was precipitated and resuspended in RNAse free water. For annealingof ssRNAs to form dsRNAs, ssRNAs were combined, heated to 95° for twominutes then allowed to cool from 70° to room temperature over 1.5-2.5hours.

[0118] DsRNA was injected into the body cavity of 15-20 young adult C.elegans hermaphrodites. Worms were typically immobilized on an agarosepad and injected with 2-5 μl of dsRNA at a concentration of 1 mg/ml.Injections were performed with visual observation using a Zeiss Axiovertcompound microscope equipped with 10× and 40× DIC objectives, forexample. Needles for microinjection were prepared using a Narishigeneedle puller, stage micromanipulator (Leitz) and a N₂-powered injector(Narishige) set at 10-20 p.s.i. After injection, 200 μl of recoverybuffer (0.1% salmon sperm DNA, 4% glucose, 2.4 mM KCl, 66 mM NaCl, 3 mMCaCl₂, 3 mM HEPES, pH 7.2) were added to the agarose pad and the wormswere allowed to recover on the agarose pad for 0.5-4 hours. Afterrecovery, the worms were transferred to NGM agar plates seeded with alawn of E. coli strain OP50 as a food source. The following day and for3 successive days thereafter, 7 individual healthy injected worms weretransferred to new NGM plates seeded with OP50. The number of eggs laidper worm per day and the number of those eggs that hatch and reachfertile adulthood were determined. As a control, Green FluorescentProtein (GFP) dsRNA was produced and injected using similar methods. GFPis a commonly used reporter gene originally isolated from jellyfish andis widely used in both prokaryotic and eukaryotic systems. The GFP geneis not present in the wild-type C. elegans genome and, therefore, GFPdsRNA does not trigger an RNAi phenotype in wild-type C. elegans. The C.elegans Δ12 FAT RNAi injection phenotype presented as a strongly reducedFl hatch-rate, with the few surviving individuals arrested in an earlylarval stage.

[0119] RNAi by feeding: C. elegans can be grown on lawns of E. coligenetically engineered to produce double stranded RNA (dsRNA) designedto inhibit Al 2 FAT expression. Briefly, E. coli were transformed with agenomic fragment of a portion of the C. elegans Δ12 FAT gene sequence,specifically a fragment 651 nucleotides long, containing the entirefirst exon and terminating just before the conserved intron splicejunction between the first exon and first intron. This construct encodesapproximately the first 217 amino acids of the C. elegans Δ12 FAT gene.The 651 nucleotide genomic fragment was cloned into an E. coliexpression vector between opposing T7 polymerase promoters. The clonewas then transformed into a strain of E. coli that carries anIPTG-inducible T7 polymerase. As a control, E. coli was transformed witha gene encoding the Green Fluorescent Protein (GFP). Feeding RNAi wasinitiated from C. elegans eggs or from C. elegans L4s. When feeding RNAiwas started from C. elegans eggs at 23° C. on NGM plates containing IPTGand E. coli expressing the C. elegans Δ12 FAT or GFP dsRNA, the C.elegans Δ12 FAT RNAi feeding phenotype presented as partially sterile F1individuals and dead F2 embryos. When feeding RNAi was started from C.elegans L4 larvae at 23° C. on NGM plates containing IPTG and E. coliexpressing the C. elegans Δ12 FAT or GFP dsRNA, the C. elegans RNAifeeding phenotype presented as partially sterile P₀ individuals withdevelopmentally arrested, sterile F1 nematodes.

[0120]C. elegans cultures grown in the presence of E. coli expressingdsRNA and those injected with dsRNA from the Δ12 FAT gene were stronglyimpaired indicating that the fatty acid desaturase-like gene provides anessential function in nematodes and that dsRNA from the fatty aciddesaturase-like gene is lethal when ingested by or injected into C.elegans.

[0121] Rescue of C. elegans Δ12 FAT RNAi Feeding Phenotype by LinoleicAcid Methyl Ester

[0122] The C. elegans Δ12 fatty acid desaturase (Fat2 protein) convertsthe mono-unsaturated oleic acid to the di-unsaturated fatty acidlinoleic acid. The Δ12 FAT RNAi prevents expression of the Δ12 fattyacid desaturase, which is predicted to cause a decrease in levels oflinoleic acid in the nematode, leading to arrested development anddeath. Addition of 3 mM linoleic acid methyl ester to the NGM media usedfor the RNAi experiment brings about a partial rescue of the Δ12 FATRNAi feeding phenotype. Addition of 3 mM oleic acid methyl ester doesnot rescue the Δ12 FAT RNAi feeding phenotype (see Table 3 below). TABLE3 C. elegans Δ12 FAT RNAi feeding phenotypes (starting with C. elegansL4 larvae as the P₀ animal) Fatty Acid Added P₀ phenotype F1 phenotypeF2 phenotype None Severely reduced Developmentally NA egg layingarrested and sterile (almost sterile) Oleic Acid Severely reducedDevelopmentally NA Methyl Ester egg laying arrested and sterile (almoststerile) Linoleic Reduced egg Moderately delayed Slightly delayed Acidlaying development and development Methyl Ester moderately reduced egglaying

[0123] Inhibitor Studies

[0124] Vernolic acid and ricinoleic acid are naturally occurringplant-produced fatty acid homologs that we predict to be specificinhibitors of Δ12 FAT enzymes. The addition of these compounds to livingcultures of C. elegans is expected to mimic the effects of the Δ12 FATRNAi experiments since, in each case, the phenotype observed shouldderive from the inhibition of the nematode Δ12 FAT. To explore thispossibility, C. elegans cultures were started from eggs on NGM platescontaining their E. coli food source and one of either 3 mM ricinoleicacid methyl ester or 3 mM vernolic acid methyl ester. Total eggs layedand hatch-rates of F1 and F2 individuals were followed and compared tonematode cultures grown in the presence of control fatty acids oleicacid methyl ester and linoleic acid methyl ester. C. elegans L4 larvaewere added to NGM plates containing OP50 E. coli and one of thefollowing methyl esters: oleic acid, linoleic acid, ricinoleic acid,vernolic acid or none. C. elegans L4 larvae growing on plates containingricinoleic acid (FIG. 11) or vernolic acid (FIG. 12) methyl estersdeveloped to mature adults more slowly than those on control platescontaining oleic acid (FIG. 9) or linoleic acid (FIG. 10) and producedvery few embryos (eggs). Of the embryos that hatched, the young larvaedisplayed severe arrested phenotypes and did not develop to adults(FIGS. 11 and 12). C. elegans cultures growing on plates containing nofatty acid methyl esters or oleic acid or linoleic acid methyl estersexhibited no dramatic lifecycle impairments (FIGS. 9 and 10).

[0125] Identification of Additional Fatty Acid Desaturase-Like Sequences

[0126] A skilled artisan can utilize the methods provided in the exampleabove to identify additional nematode fatty acid desaturase-likesequences, e.g., fatty acid desaturase-like sequences (including Δ12fatty acid desaturase sequences) from nematodes other than M. incognita,H. glycines, D. immitis, S. stercoralis, R. axei and/or C. elegans. Inaddition, nematode fatty acid desaturase-like sequences can beidentified by a variety of methods including computer-based databasesearches, hybridization-based methods, and functional complementation.

[0127] Database Identification A nematode fatty acid desaturase-likesequence can be identified from a sequence database, e.g., a protein ornucleic acid database using a sequence disclosed herein as a query.Sequence comparison programs can be used to compare and analyze thenucleotide or amino acid sequences. One such software package is theBLAST suite of programs from the National Center for BiotechnologyInstitute (NCBI; Altschul et al. (1997) Nuc. Acids Research25:3389-3402). A fatty acid desaturase-like sequence of the inventioncan be used to query a sequence database, such as nr, dbest (expressedsequence tag (EST) sequences), and htgs (high-throughput genomesequences), using a computer-based search, e.g., FASTA, BLAST, orPSI-BLAST search. Homologous sequences in other species (e.g., humansand animals) can be detected in a PSI-BLAST search of a database such asnr (E value=10, H value=1e-2, using, for example, four iterations;http://www.ncbi.nlm.nih.gov/). Sequences so obtained can be used toconstruct a multiple alignment, e.g., a ClustalX alignment, and/or tobuild a phylogenetic tree, e.g., in ClustalX using the Neighbor-Joiningmethod (Saitou et al. (1987) Mol. Biol. Evol. 4:406-425) andbootstrapping (1000 replicates; Felsenstein (1985) Evolution39:783-791). Distances maybe corrected for the occurrence of multiplesubstitutions [D_(COIT)=−ln(1−D−D²/5) where D is the fraction of aminoacid differences between two sequences] (Kimura (1983) The NeutralTheory of Molecular Evolution, Cambridge University Press).

[0128] The aforementioned search strategy can be used to identify fattyacid desaturase-like sequences in nematodes of the followingnon-limiting, exemplary genera:

[0129] Plant Parasitic Nematode Genera Include:

[0130] Afrina, Anguina, Aphelenchoides, Belonolaimus, Bursaphelenchus,Cacopaurus, Cactodera, Criconema, Criconemoides, Cryphodera,Ditylenchus, Dolichodorus, Dorylaimus, Globodera, Helicotylenchus,Hemicriconemoides, Hemicycliophora, Heterodera, Hirschmanniella,Hoplolaimus, Hypsoperine, Longidorus, Meloidogyne, Mesoanguina,Nacobbus, Nacobbodera, Panagrellus, Paratrichodorus, Paratylenchus,Pratylenchus, Pterotylench us, Punctodera, Radopholus,Rhadinaphelenchus, Rotylenchulus, Rotylenchus, Scutellonema, Subanguina,Thecavermiculatus, Trichodorus, Turbatrix, Tylenchorhynchus,Tylenchulus, Xiphinema.

[0131] Animal and Human Parasitic Nematode Genera Include:

[0132] Acanthocheilonema, Aelurostrongylus, Ancylostoma,Angiostrongylus, Anisakis, Ascaris, Ascarops, Bunostomum, Brugia,Capillaria, Chabertia, Cooperia, Crenosoma, Cyathostome species (SmallStrongyles), Dictyocaulus, Dioctophyma, Dipetalonema, Dirofiliaria,Dracunculus, Draschia, Elaneophora, Enterobius, Filaroides, Gnathostoma,Gonylonema, Habronema, Haemonchus, Hyostrongylus, Lagochilascaris,Litomosoides, Loa, Mammomonogamus, Mansonella, Muellerius,Metastrongylid, Necator, Nematodirus, Nippostrongylus, Oesophagostomum,Ollulanus, Onchocerca, Ostertagia, Oxyspirura, Oxyuris, Parafilaria,Parascaris, Parastrongyloides, Parelaphostrongylus, Physaloptera,Physocephalus, Protostrongylus, Pseudoterranova, Setaria, Spirocerca,Stephanurus, Stephanofilaria, Strongyloides, Strongylus, Spirocerca,Syngamus, Teladorsagia, Thelazia, Toxascaris, Toxocara, Trichinella,Trichostrongylus, Trichuris, Uncinaria, and Wuchereria.

[0133] Particularly Preferred Nematode Genera Include:

[0134] Plant parasitic: Anguina, Aphelenchoides, Belonolaimus,Bursaphelenchus, Ditylenchus, Dolichodorus, Globodera, Heterodera,Hoplolaimus, Longidorus, Meloidogyne, Nacobbus, Pratylenchus,Radopholus, Rotylenchus, Tylenchulus, Xiphinema.

[0135] Animal & Human parasitic: Ancylostoma, Ascaris, Brugia,Capillaria, Cooperia, Cyathostome species, Dictyocaulus, Dirofiliaria,Dracunculus, Enterobius, Haemonchus, Necator, Nematodirus,Oesophagostomum, Onchocerca, Ostertagia, Oxyspirura, Oxyuris,Parascaris, Strongyloides, Strongylus, Syngamus, Teladorsagia, Thelazia,Toxocara, Trichinella, Trichostrongylus, Trichuris, and Wuchereria.

[0136] Particularly Preferred Nematode Species Include:

[0137] Plant parasitic: Anguina tritici, Aphelenchoides fragariae,Belonolaimus longicaudatus, Bursaphelenchus xylophilus, Ditylenchusdestructor, Ditylenchus dipsaci, Dolichodorus heterocephalous, Globoderapallida, Globodera rostochiensis, Globodera tabacum, Heterodera avenae,Heterodera cardiolata, Heterodera carotae, Heterodera cruciferae,Heterodera glycines, Heterodera major, Heterodera schachtii, Heteroderazeae, Hoplolaimus tylenchiformis, Longidorus sylphus, Meloidogyneacronea, Meloidogyne arenaria, Meloidogyne chitwoodi, Meloidogyneexigua, Meloidogyne graminicola, Meloidogyne hapla, Meloidogyneincognita, Meloiclogynejavanica, Meloidogyne nassi, Nacobbusbatatiformis, Pratylenchus brachyurus, Pratylenchus coffeae,Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus zeae,Radopholus similis, Rotylenchus reniformis, Tylenchulus semipenetrans,Xiphinema americanum.

[0138] Animal & Human parasitic: Ancylostoma braziliense, Ancylostomacaninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostomatubaeforme, Ascaris suum, Ascaris lumbrichoides, Brugia malayi,Capillaria bovis, Capillaria plica, Capillaria feliscati, Cooperiaoncophora, Cooperia punctata, Cyathostome species, Dictyocaulus filaria,Dictyocaulus viviparus, Dictyocaulus arnfieldi, Dirofiliaria immitis,Dracunculus insignis, Enterobius vermicularis, Haemonchus contortus,Haemonchus placei, Necator americanus, Nematodirus helvetianus,Oesophagostomum racliatum, Onchocerca volvulus, Onchocerca cervicalis,Ostertagia ostertagi, Ostertagia circumcincta, Oxyuris equi, Parascarisequorum, Strongyloides stercoralis, Strongylus vulgaris, Strongylusedentatus, Syngamus trachea, Teladorsagia circumcincta, Toxocara cati,Trichinella spiralis, Trichostrongylus axei, Trichostrongyluscolubriformis, Trichuris vulpis, Trichuris suis, Trichurs trichiura, andWuchereria bancrofti.

[0139] Further, a fatty acid desaturase-like sequence can be used toidentify additional fatty acid desaturase-like sequence homologs withina genome. Multiple homologous copies of a fatty acid desaturase-likesequence can be present. For example, a nematode fatty aciddesaturase-like sequence can be used as a seed sequence in an iterativePSI-BLAST search (default parameters, substitution matrix=Blosum62, gapopen=11, gap extend=1) of a nonredundant database such as wormpep (Evalue=1e-2, H value=1e-4, using, for example 4 iterations) to determinethe number of homologs in a database, e.g., in a database containing thecomplete genome of an organism. A nematode fatty acid desaturase-likesequence can be present in a genome along with 1, 2, 3, 4, 5, 6, 8, 10,or more homologs.

[0140] Hybridization Methods A nematode fatty acid desaturase-likesequence can be identified by a hybridization-based method using asequence provided herein as a probe. For example, a library of nematodegenomic or cDNA clones can be hybridized under low stringency conditionswith the probe nucleic acid. Stringency conditions can be modulated toreduce background signal and increase signal from potential positives.Clones so identified can be sequenced to verify that they encode fattyacid desaturase-like sequences.

[0141] Another hybridization-based method utilizes an amplificationreaction (e.g., the polymerase chain reaction (PCR)). Oligonucleotides,e.g., degenerate oligonucleotides, are designed to hybridize to aconserved region of a fatty acid desaturase-like sequence. Theoligonucleotides are used as primers to amplify a fatty aciddesaturase-like sequence from template nucleic acid from a nematode,e.g., a nematode other than M. incognita, and/or C. elegans. Theamplified fragment can be cloned and/or sequenced.

[0142] Complementation Methods A nematode fatty acid desaturase-likesequence can be identified from a complementation screen for a nucleicacid molecule that restores fatty acid desaturase-like activity to acell lacking a fatty acid desaturase-like activity. Routine methods canbe used to construct strains (i.e., nematode, yeast, bacterial strains)that lack fatty acid desaturase activity. For example, a nematode strainmutated at the fatty acid desaturase gene locus can be grown (i.e.,rescued) on supplements such as Δ12 unsaturated fatty acids. Such astrain can be transformed with nematode cDNAs predicted to encode fattyacid desaturases. Strains can be identified in which fatty aciddesaturase activity is restored by selecting for those transgenic linesthat exhibit growth in the absensce of supplemental Δ12 unsaturatedfatty acids. The plasmid harbored by the rescued strain can be recoveredto identify and/or characterize the inserted nematode cDNA that providesfatty acid desaturase-like activity when expressed. Similarly, abacterial and/or yeast strain can be used as a selection system, wherebythe Δ12 fatty acid desaturase gene(s) can be mutated using, for example,phage transduction (Clark et al. (1983) Biochem. 22:5897-5902; Simon etal. (1980) J. Bacteriology 142:621-632). The mutant cell line can besustained on exongenous unsaturated fatty acids. A strain lacking Δ12fatty acid desaturase gene(s) can be transformed with a plasmid libraryexpressing nematode cDNAs. Strains can be identified in which fatty aciddesaturase activity is restored, i.e., that can grow in the absence ofexogenous fatty acids. In still another embodiment, a microorganism(i.e., a yeast strain) that naturally does not contain a Δ12 fatty aciddesaturase can be transformed with plasmids expressing nematode genes.Transformed strains that exhibit Δ12 fatty acid desaturase activity canbe identified using, GC analysis to measure fatty acid composition.Those clones that convert oleic acid to linoleic acid can be selected,for example (Sakurdani et al. (1999) Eur. J. Biochem. 261:812-820.

[0143] Full-length cDNA and Sequencing Methods The following methods canbe used, e.g., alone or in combination with another method describedherein, to obtain full-length nematode fatty acid desaturase-like genesand determine their sequences.

[0144] Plant parasitic nematodes are maintained on greenhouse potcultures depending on nematode preference. Root Knot Nematodes(Meloidogyne sp) are propagated on Rutgers tomato (Burpee), whileSoybean Cyst Nematodes (Heterodera sp) are propagated on soybean. Totalnematode RNA is isolated using the TRIZOL reagent (Gibco BRL). Briefly,2 ml of packed worms are combined with 8 ml TRIZOL reagent andsolubilized by vortexing. Following 5 minutes of incubation at roomtemperature, the samples are divided into smaller volumes and spun at14,000×g for 10 minutes at 4° C. to remove insoluble material. Theliquid phase is extracted with 200 μl of chloroform, and the upperaqueous phase is removed to a fresh tube. The RNA is precipitated by theaddition of 500 μl of isopropanol and centrifuged to pellet. The aqueousphase is carefully removed, and the pellet is washed in 75% ethanol andspun to re-collect the RNA pellet. The supernatant is carefully removed,and the pellet is air dried for 10 minutes. The RNA pellet isresuspended in 50 μl of DEPC-H₂O and analyzed by spectrophotometry at λ260 and 280 nm to determine yield and purity. Yields can be 1-4 mg oftotal RNA from 2 ml of packed worms.

[0145] Full-length cDNAs can be generated using 5′ and 3′ RACEtechniques in combination with EST sequence information. The moleculartechnique 5′ RACE (Life Technologies, Inc., Rockville, Md.) can beemployed to obtain complete or near-complete 5′ ends of cDNA sequencesfor nematode fatty acid desaturase-like cDNA sequences. Briefly,following the instructions provided by Life Technologies, first strandcDNA is synthesized from total nematode RNA using Murine Leukemia VirusReverse Transcriptase (M-MLV RT) and a gene specific “antisense” primer,e.g., designed from available EST sequence. RNase H is used to degradethe original mRNA template. The first strand cDNA is separated fromunincorporated dNTPs, primers, and proteins using a GlassMAX SpinCartridge. Terminal deoxynucleotidyl transferase (TdT) is used togenerate a homopolymeric dC tailed extension by the sequential additionof dCTP nucleotides to the 3′ end of the first strand cDNA. Followingaddition of the dC homopolymeric extension, the first strand cDNA isdirectly amplified without further purification using Taq DNApolymerase, a gene specific “antisense” primer designed from availableEST sequences to anneal to a site located within the first strand cDNAmolecule, and a deoxyinosine-containing primer that anneals to thehomopolymeric dC tailed region of the cDNA in a polymerase chainreaction (PCR). 5′ RACE PCR amplification products are cloned into asuitable vector for further analysis and sequencing.

[0146] The molecular technique, 3′ RACE (Life Technologies, Inc.,Rockville, Md.), can be employed to obtain complete or near-complete 3′ends of cDNA sequences for nematode fatty acid desaturase-like cDNAsequences. Briefly, following the instructions provided by LifeTechnologies (Rockville, Md.), first strand cDNA synthesis is performedon total nematode RNA using SuperScript™ Reverse Transcriptase and anoligo-dT primer that anneals to the polyA tail. Following degradation ofthe original mRNA template with RNase H, the first strand cDNA isdirectly PCR amplified without further purification using Taq DNApolymerase, a gene specific primer designed from available EST sequencesto anneal to a site located within the first strand cDNA molecule, and a“universal” primer which contains sequence identity to 5′ end of theoligo-dT primer. 3′ RACE PCR amplification products are cloned into asuitable vector for further analysis and sequencing.

[0147] Nucleic Acid Variants

[0148] Isolated nucleic acid molecules of the present invention includenucleic acid molecules that have an open reading frame encoding a fattyacid desaturase-like polypeptide. Such nucleic acid molecules includemolecules having: the sequences recited in SEQ ID NO: 1, 2, 3, 4, 5, 6and/or 7; and sequences coding for the fatty acid desaturase-likeproteins recited in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14. Thesenucleic acid molecules can be used, for example, in a hybridizationassay to detect the presence of a M. incognita, H. glycines, D. immitis,S. stercoralis or R. axei nucleic acid in a sample.

[0149] The present invention includes nucleic acid molecules such asthose shown in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 that may besubjected to mutagenesis to produce single or multiple nucleotidesubstitutions, deletions, or insertions. Nucleotide insertionalderivatives of the nematode gene of the present invention include 5′ and3′ terminal fusions as well as intra-sequence insertions of single ormultiple nucleotides. Insertional nucleotide sequence variants are thosein which one or more nucleotides are introduced into a predeterminedsite in the nucleotide sequence, although random insertion is alsopossible with suitable screening of the resulting product. Deletionvariants are characterized by the removal of one or more nucleotidesfrom the sequence. Nucleotide substitution variants are those in whichat least one nucleotide in the sequence has been removed and a differentnucleotide inserted in its place. Such a substitution may be silent(e.g., synonymous), meaning that the substitution does not alter theamino acid defined by the codon. Alternatively, substitutions aredesigned to alter one amino acid for another amino acid (e.g.,non-synonymous). A non-synonymous substitution can be conservative ornon-conservative. A substitution can be such that activity, e.g., fattyacid desaturase-like activity, is not impaired. A conservative aminoacid substitution results in the alteration of an amino acid for asimilar acting amino acid, or amino acid of like charge, polarity, orhydrophobicity, e.g., an amino acid substitution listed in Table 3below. At some positions, even conservative amino acid substitutions candisrupt the activity of the polypeptide. TABLE 4 Conservative Amino AcidReplacements For Amino Acid Code Replace with any of . . . Alanine AlaGly, Cys, Ser Arginine Arg Lys, His Asparagine Asn Asp, Glu, Gln,Aspartic Acid Asp Asn, Glu, Gln Cysteine Cys Met, Thr, Ser Glutamine GlnAsn, Glu, Asp Glutamic Acid Glu Asp, Asn, Gln Glycine Gly Ala HistidineHis Lys, Arg Isoleucine Ile Val, Leu, Met Leucine Leu Val, Ile, MetLysine Lys Arg, His Methionine Met Ile, Leu, Val Phenylalanine Phe Tyr,His, Trp Proline Pro Serine Ser Thr, Cys, Ala Threonine Thr Ser, Met,Val Tryptophan Trp Phe, Tyr Tyrosine Tyr Phe, His Valine Val Leu, Ile,Met

[0150] The current invention also embodies splice variants of nematodefatty acid desaturase-like sequences.

[0151] Another aspect of the present invention embodies apolypeptide-encoding nucleic acid molecule that is capable ofhybridizing under conditions of low stringency (or high stringency) tothe nucleic acid molecule put forth in SEQ ID NO: 1, 2, 3, 4, 5, 6and/or 7 or their complements.

[0152] The nucleic acid molecules that encode for fatty aciddesaturase-like polypeptides may correspond to the naturally occurringnucleic acid molecules or may differ by one or more nucleotidesubstitutions, deletions, insertions, and/or additions. Thus, thepresent invention extends to genes and any functional mutants,derivatives, parts, fragments, naturally occurring polymorphisms,homologs or analogs thereof or non-functional molecules. Such nucleicacid molecules can be used to detect polymorphisms of fatty aciddesaturase genes or fatty acid desaturase-like genes, e.g., in othernematodes. As mentioned below, such molecules are useful as geneticprobes; primer sequences in the enzymatic or chemical synthesis of thegene; or in the generation of immunologically interactive recombinantmolecules. Using the information provided herein, such as the nucleotidesequence SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7, a nucleic acid moleculeencoding a fatty acid desaturase-like molecule may be obtained usingstandard cloning and a screening techniques, such as a method describedherein.

[0153] Nucleic acid molecules of the present invention can be in theform of RNA, such as mRNA, or in the form of DNA, including, forexample, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded. Thenucleic acids may be in the form of RNA/DNA hybrids. Single-stranded DNAor RNA can be the coding strand, also referred to as the sense strand,or the non-coding strand, also known as the anti-sense strand.

[0154] Expression of Fatty Acid Desaturase-Like Polypeptides

[0155] One embodiment of the present invention includes a recombinantnucleic acid molecule, which includes at least one isolated nucleic acidmolecule depicted in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7, inserted in avector capable of delivering and maintaining the nucleic acid moleculeinto a cell. The DNA molecule may be inserted into an autonomouslyreplicating factor (suitable vectors include, for example, pGEM3Z andpcDNA3, and derivatives thereof). The vector nucleic acid may be abacteriophage DNA such as bacteriophage lambda or M13 and derivativesthereof. The vector may be either RNA or DNA, single- ordouble-stranded, prokaryotic, eukaryotic, or viral. Vectors can includetransposons, viral vectors, episomes, (e.g., plasmids), chromosomesinserts, and artificial chromosomes (e.g. BACs or YACs). Construction ofa vector containing a nucleic acid described herein can be followed bytransformation of a host cell such as a bacterium. Suitable bacterialhosts include, but are not limited to, E. coli. Suitable eukaryotichosts include yeast such as S. cerevisiae, other fungi, vertebratecells, invertebrate cells (e.g., insect cells), plant cells, humancells, human tissue cells, and whole eukaryotic organisms. (e.g., atransgenic plant or a transgenic animal). Further, the vector nucleicacid can be used to generate a virus such as vaccinia or baculovirus.

[0156] The present invention also extends to genetic constructs designedfor polypeptide expression. Generally, the genetic construct alsoincludes, in addition to the encoding nucleic acid molecule, elementsthat allow expression, such as a promoter and regulatory sequences. Theexpression vectors may contain transcriptional control sequences thatcontrol transcriptional initiation, such as promoter, enhancer,operator, and repressor sequences. A variety of transcriptional controlsequences are well known to those in the art and may be functional in,but are not limited to, a bacterium, yeast, plant, or animal cell. Theexpression vector can also include a translation regulatory sequence(e.g., an untranslated 5′ sequence, an untranslated 3′ sequence, a polyA addition site, or an internal ribosome entry site), a splicingsequence or splicing regulatory sequence, and a transcriptiontermination sequence. The vector can be capable of autonomousreplication or it can integrate into host DNA.

[0157] In an alternative embodiment, the DNA molecule is fused to areporter gene such as β-glucuronidase gene, β-galactosidase (lacZ),chloramphenicol-acetyltransferase gene, a gene encoding greenfluorescent protein (and variants thereof), or red fluorescent proteinfirefly luciferase gene, among others. The DNA molecule can also befused to a nucleic acid encoding a polypeptide affinity tag, e.g.glutathione S-transferase (GST), maltose E binding protein, protein A,FLAG tag, hexa-histidine, or the influenza HA tag. The affinity tag orreporter fusion joins the reading frames of SEQ ID NO: 1, 2, 3, 4, 5, 6and/or 7 to the reading frame of the reporter gene encoding the affinitytag such that a translational fusion is generated. Expression of thefusion gene results in translation of a single polypeptide that includesboth a nematode fatty acid desaturase-like region and a reporter proteinor affinity tag. The fusion can also join a fragment of the readingframe of SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7. The fragment can encode afunctional region of the fatty acid desaturase-like polypeptides, astructurally intact domain, or an epitope (e.g., a peptide of about 8,10, 20, or 30 or more amino acids). A nematode fatty aciddesaturase-like nucleic acid that includes at least one of a regulatoryregion (e.g., a 5′ regulatory region, a promoter, an enhancer, a 5′untranslated region, a translational start site, a 3′ untranslatedregion, a polyadenylation site, or a 3′ regulatory region) can also befused to a heterologous nucleic acid. For example, the promoter of afatty acid desaturase-like nucleic acid can be fused to a heterologousnucleic acid, e.g., a nucleic acid encoding a reporter protein.

[0158] Suitable cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Atransformed cell of the present invention is also herein referred to asa recombinant or transgenic cell. Suitable cells can either beuntransformed cells or cells that have already been transformed with atleast one nucleic acid molecule. Suitable cells for transformationaccording to the present invention can either be: (i) endogenouslycapable of expressing the fatty acid desaturase-like protein or; (ii)capable of producing such protein after transformation with at least onenucleic acid molecule of the present invention.

[0159] In an exemplary embodiment, a nucleic acid of the invention isused to generate a transgenic nematode strain, e.g., a transgenic C.elegans strain. To generate such a strain, nucleic acid linked to a C.elegans promoter is injected into the gonad of a nematode, thusgenerating a heritable extrachromosomal array containing the nucleicacid (see, e.g., Mello et al. (1991) EMBO J. 10:3959-3970). Thetransgenic nematode can be propagated to generate a strain harboring thetransgene. To identify specific inhibitors of the M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei Δ12 FAT2, the C. elegansΔ12 FAT gene can be “knocked out” by continuous growth on E. coliengineered to produce dsRNA homologous to the C. elegans Δ12 FAT gene(described earlier). Nematodes of the strain can be used in screens toidentify inhibitors specific for a M. incognita, H. glycines, D.immitis, S. stercoralis or R. axei fatty acid desaturase-like gene.

[0160] In another embodiment, a nucleic acid of the invention can becloned behind a yeast-specific transcription promoter can be used togenerate a transgenic yeast strain, such as Saccharomyces cerevisiae.The S. cerevisiae strain can be transformed using the lithium acetateprocedure (Ito et al. (1983) J. Bacteriology 153:163-168; Sakuradani(1999) Eur J. Biochem. 261:812-820). Such a strain can be used toidentify inhibitors specific for a M. incognita, H. glycines, D.immitis, S. stercoralis or R. axei fatty acid desaturase-likepolypeptide.

[0161] Production of Fatty Acid Desaturase-Like Polypeptide Substratesand Inhibitors

[0162] In still another embodiment, a nucleic acid of the invention canbe used to generate a transgenic plant such as Arabidopis thaliana, amodel legume Medicago truncatula, or any plant of interest, e.g., anematode host. For example, the fatty acid desaturase like-gene can becloned into a vector under the control of the cauliflower mosaic virus(CaMV) 35S promoter/nopaline synthase terminator cassette (Baulcombe etal. (1986) Nature 321:446-449) which can then be introduced into anAgrobacterium strain by the freeze thaw method.

[0163] Agrobacterium-mediated transformation can be accomplished by theplanta-vacuum-infiltration method (Bouchez et al. (1993) C.R. Acad. Sci.Paris 316:1188-1193) and transformed transgeneic plant lines can beselected (Spychalla et al. (1997) Proc. Natl. Acad. Sci. 94:1142-1147).Such a plant line can be used to identify inhibitors specific for M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like polypeptides or other plant fatty acid desaturase-likepolypeptides. Fatty acid desaturase can be expressed in soybean and/orsoybean somatic embryos using, for example, particle bombardment methodof transformation (Finer et al. (1991) In Vitro Cell. Dev. Biol.27:175-182; Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96:12935-12940). It is also desirable to generate plants with increasedresistance to inhibitors of fatty acid desaturase-like polypeptides byproviding the plant with a transgene expressing a fatty acid desaturase.A transformed cell that harbors a M. incognita fatty acid desaturasepolypeptide may naturally produce products of Δ12 fatty acid desaturases(e.g. linoleic acid). In this circumstance, the cell may produce ahigher proportion of Δ2-desaturated fatty acids than an otherwisesimilar cell lacking the M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase polypeptide.

[0164] If the host cell does not naturally produce a substrate for fattyacid desaturase, one or more substrates can be provided exogenously tocells transformed with an expressible fatty acid desaturasepolynucleotide (e.g., by topical application (Spychalla et al. (1997)Proc. Natl. Acad. Sci. USA 94:1142-1147)), or fatty acid desaturase canbe co-expressed in cells together with one or more cloned genes thatencode polypeptides that can produce substrate compounds from precursorcompounds in such cells.

[0165] A transformed cell may also be engineered that harbors apolypeptide that produces inhibitors of the Δ12 fatty aciddesaturase-like gene of nematodes. For example, a cell may be engineeredto produce a Δ12 hydroxylase, a Δ12 acetylenase, and/or a Δ12epoxygenase. Such polypeptides may produce fatty acid analogs thatinhibit the nematode Δ12 fatty acid desaturase-like polypeptide (i.e.,ricinoleic acid, crepenynic acid, and vernolic acid). Such genes may belinked to a root-specific promoter and transformed into a plant, forexample. Examples of suitable genes include: Crepsis palaestina Δ12fatty acid epoxygenase (GenBank® Accession No. CAA76156; producesvernolic acid); Crepis alpina Δ12 fatty acid acetylenase (GenBank®Accession No. CAA76158; produces crepenynic acid); Ricinus Communisoleate 12-hydroxylase (GenBank® Accession No. AAC49010; producesricinoleic acid); Momordica charantia Δ12 oleic desaturase-like protein(GenBank® Accession No. AAF05916; produces alpha-eleostearic acid); andImpatiens balsamina Δ12 oleic acid desaturase-like protein (GenBank®Accession No. AAF05915; produces alpha-eleostearic acid).

[0166] Oligonucleotides

[0167] Also provided are oligonucleotides that can form stable hybridswith a nucleic acid molecule of the present invention. Theoligonucleotides can be about 10 to 200 nucleotides, about 15 to 120nucleotides, or about 17 to 80 nucleotides in length, e.g., about 10,20, 30, 40, 50, 60, 80, 100, 120 nucleotides in length. Theoligonucleotides can be used as probes to identify nucleic acidmolecules, primers to produce nucleic acid molecules, or therapeuticreagents to inhibit nematode fatty acid desaturase-like protein activityor production (e.g., antisense, triplex formation, ribozyme, and/or RNAdrug-based reagents). The present invention includes oligonucleotides ofRNA (ssRNA and dsRNA), DNA, or derivatives of either. The inventionextends to the use of such oligonucleotides to protect non-nematodeorganisms (for example e.g., plants and animals) from disease byreducing the viability of infecting namatodes, e.g., using a technologydescribed herein. Appropriate oligonucleotide-containing therapeuticcompositions can be administered to a non-nematode organism usingtechniques known to those skilled in the art, including, but not limitedto, transgenic expression in plants or animals.

[0168] Primer sequences can be used to amplify a fatty aciddesaturase-like nucleic acid or fragment thereof. For example, at least10 cycles of PCR amplification can be used to obtain such an amplifiednucleic acid. Primers can be at least about 8-40, 10-30 or 14-25nucleotides in length, and can anneal to a nucleic acid “templatemolecule”, e.g., a template molecule encoding a fatty aciddesaturase-like genetic sequence, or a functional part thereof, or itscomplementary sequence. The nucleic acid primer molecule can be anynucleotide sequence of at least 10 nucleotides in length derived from,or contained within sequences depicted in SEQ ID NO: 1, 2, 3, 4, 5, 6and/or 7 and their complements. The nucleic acid template molecule maybe in a recombinant form, in a virus particle, bacteriophage particle,yeast cell, animal cell, plant cell, fungal cell, or bacterial cell. Aprimer can be chemically synthesized by routine methods.

[0169] This invention embodies any fatty acid desaturase-like sequencesthat are used to identify and isolate similar genes from otherorganisms, including nematodes, prokaryotic organisms, and othereukaryotic organisms, such as other animals and/or plants.

[0170] In another embodiment, the invention provides oligonucleotidesthat are specific for a M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase-like nucleic acid molecule.Such oligonucleotides can be used in a PCR test to determine if a M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei nucleicacid is present in a sample, e.g., to monitor a disease caused M.incognita, H. glycines, D. immitis or S. stercoralis.

[0171] Protein Production

[0172] Isolated fatty acid desaturase-like proteins from nematodes canbe produced in a number of ways, including production and recovery ofthe recombinant proteins and/or chemical synthesis of the protein. Inone embodiment, an isolated nematode fatty acid desaturase-like proteinis produced by culturing a cell, e.g., a bacterial, fungal, plant, oranimal cell, capable of expressing the protein, under conditions foreffective production and recovery of the protein. The nucleic acid canbe operably linked to a heterologous promoter, e.g., an induciblepromoter or a constitutive promoter. Effective growth conditions aretypically, but not necessarily, in liquid media comprising salts, water,carbon, nitrogen, phosphate sources, minerals, and other nutrients, butmay be any solution in which fatty acid desaturase-like proteins may beproduced.

[0173] In one embodiment, recovery of the protein may refer tocollecting the growth solution and need not involve additional steps ofpurification. Proteins of the present invention, however, can bepurified using standard purification techniques, such as, but notlimited to, affinity chromatography, thermaprecipitation, immunoaffinitychromatography, ammonium sulfate precipitation, ion exchangechromatography, filtration, electrophoresis, hydrophobic interactionchromatography, and others.

[0174] The fatty acid desaturase-like polypeptide can be fused to anaffinity tag, e.g., a purification handle (e.g.,glutathione-S-reductase, hexa-histidine, maltose binding protein,dihydrofolate reductases, or chitin binding protein) or an epitope tag(e.g., c-myc epitope tag, FLAG™ tag, or influenza HA tag). Affinitytagged and epitope tagged proteins can be purified using routineart-known methods.

[0175] Antibodies Against Fatty Acid Desaturase-Like Polypeptides

[0176] Recombinant fatty acid desaturase-like gene products orderivatives thereof can be used to produce immunologically interactivemolecules, such as antibodies, or functional derivatives thereof. Usefulantibodies include those that bind to a polypeptide that hassubstantially the same sequence as the amino acid sequences recited inSEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14, or that has at least 80%similarity over 50 or more amino acids to these sequences. In apreferred embodiment, the antibody specifically binds to a polypeptidehaving the amino acid sequence recited in SEQ ID NO: 8, 9, 10, 11, 12,13 and/or 14. The antibodies can be antibody fragments and geneticallyengineered antibodies, including single chain antibodies or chimericantibodies that can bind to more than one epitope. Such antibodies maybe polyclonal or monoclonal and may be selected from naturally occurringantibodies or may be specifically raised to a recombinant fatty aciddesaturase-like protein.

[0177] Antibodies can be derived by immunization with a recombinant orpurified fatty acid desaturase-like gene or gene product. As usedherein, the term “antibody” refers to an immunoglobulin, or fragmentthereof. Examples of antibody fragments include F(ab) and F(ab′)₂fragments, particularly functional ones able to bind epitopes. Suchfragments can be generated by proteolytic cleavage, e.g., with pepsin,or by genetic engineering. Antibodies can be polyclonal, monoclonal, orrecombinant. In addition, antibodies can be modified to be chimeric, orhumanized. Further, an antibody can be coupled to a label or a toxin.

[0178] Antibodies can be generated against a full-length fatty aciddesaturase-like protein, or a fragment thereof, e.g., an antigenicpeptide. Such polypeptides can be coupled to an adjuvant to improveimmunogenicity. Polyclonal serum is produced by injection of the antigeninto a laboratory animal such as a rabbit and subsequent collection ofsera. Alternatively, the antigen is used to immunize mice. Lymphocyticcells are obtained from the mice and fused with myelomas to formhybridomas producing antibodies.

[0179] Peptides for generating fatty acid desaturase-like antibodies canbe about 8, 10, 15, 20, 30 or more amino acid residues in length, e.g.,a peptide of such length obtained from SEQ ID NO: 8, 9, 10, 11, 12, 13and/or 14. Peptides or epitopes can also be selected from regionsexposed on the surface of the protein, e.g., hydrophilic or amphipathicregions. An epitope in the vicinity of the active or binding site can beselected such that an antibody binding such an epitope would blockaccess to the active site or prevent binding. Antibodies reactive with,or specific for, any of these regions, or other regions or domainsdescribed herein are provided. An antibody to a fatty aciddesaturase-like protein can modulate a fatty acid desaturase-likeactivity.

[0180] Monoclonal antibodies, which can be produced by routine methods,are obtained in abundance and in homogenous form from hybridomas formedfrom the fusion of immortal cell lines (e.g., myelomas) with lymphocytesimmunized with fatty acid desaturase-like polypeptides such as those setforth in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14.

[0181] In addition, antibodies can be engineered, e.g., to produce asingle chain antibody (see, for example, Colcher et al. (1999) Ann N YAcad Sci 880: 263-80; and Reiter (1996) Clin Cancer Res 2: 245-52). Instill another implementation, antibodies are selected or modified basedon screening procedures, e.g., by screening antibodies or fragmentsthereof from a phage display library.

[0182] Antibodies of the present invention have a variety of importantuses within the scope of this invention. For example, such antibodiescan be used: (i) as therapeutic compounds to passively immunize ananimal in order to protect the animal from nematodes susceptible toantibody treatment; (ii) as reagents in experimental assays to detectpresence of nematodes; (iii) as tools to screen for expression of thegene product in nematodes, animals, fungi, bacteria, and plants; and/or(iv) as a purification tool of fatty acid desaturase-like protein; (v)as fatty acid desaturase inhibitors/activators that can be expressed orintroduced into plants or animals for therapeutic purposes.

[0183] An antibody against a fatty acid desaturase-like protein can beproduced in a plant cell, e.g., in a transgenic plant or in culture(see, e.g., U.S. Pat. No. 6,080,560).

[0184] Antibodies that specifically recognize a M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like proteins can be used to identify a M. incognita, H.glycines, D. iinmitis, S. stercoralis or R. axei nematodes, and, thus,can be used to monitor a disease caused by M. incognita, H. glycines, D.immitis or S. stercoralis.

[0185] Nucleic Acids Agents

[0186] Also featured are isolated nucleic acids that are antisense tonucleic acids encoding nematode fatty acid desaturase-like proteins. An“antisense” nucleic acid includes a sequence that is complementary tothe coding strand of a nucleic acid encoding a fatty aciddesaturase-like protein. The complementarity can be in a coding regionof the coding strand or in a noncoding region, e.g., a 5′ or 3′untranslated region, e.g., the translation start site. The antisensenucleic acid can be produced from a cellular promoter (e.g., a RNApolymerase II or III promoter), or can be introduced into a cell, e.g.,using a liposome. For example, the antisense nucleic acid can be asynthetic oligonucleotide having a length of about 10, 15, 20, 30, 40,50, 75, 90, 120 or more nucleotides in length.

[0187] An antisense nucleic acid can be synthesized chemically orproduced using enzymatic reagents, e.g., a ligase. An antisense nucleicacid can also incorporate modified nucleotides, and artificial backbonestructures, e.g., phosphorothioate derivative, and acridine substitutednucleotides.

[0188] Ribozymes: The antisense nucleic acid can be a ribozyme. Theribozyme can be designed to specifically cleave RNA, e.g., a fatty aciddesaturase-like mRNA. Methods for designing such ribozymes are describedin U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature334:585-591. For example, the ribozyme can be a derivative ofTetrahymena L-19 IVS RNA in which the nucleotide sequence of the activesite is modified to be complementary to a fatty acid desaturase-likenucleic acid (see, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cechet al. U.S. Pat. No. 5,116,742).

[0189] Peptide Nucleic acid (PNA): An antisense agent directed against afatty acid desaturase-like nucleic acid can be a peptide nucleic acid(PNA). See Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23)for methods and a description of the replacement of the deoxyribosephosphate backbone for a pseudopeptide backbone. A PNA can specificallyhybridize to DNA and RNA under conditions of low ionic strength as aresult of its electrostatic properties. The synthesis of PNA oligomerscan be performed using standard solid phase peptide synthesis protocolsas described in Hyrup et al. (1996) supra and Perry-O'Keefe et al. Proc.Natl. Acad. Sci. 93: 14670-675.

[0190] RNA Mediated Interference (RNAi): A double stranded RNA (dsRNA)molecule can be used to inactivate expression of a fatty aciddesaturase-like gene in a cell by a process known as RNAmediated-interference (RNAi; e.g., Fire et al. (1998) Nature391:806-811, and Gonczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of a fatty aciddesaturase-like nucleic acid described herein or a fragment thereof. Themolecule can be injected into a cell, or a syncitia, e.g., a nematodegonad as described in Fire et al., supra.

[0191] In one embodiment, dsRNA can be introduced by soaking ofnematodes. Double-stranded RNA may be introduced to nematodes bydirectly soaking individual worms in a solution of dsRNA. Soaking of C.elegans in a solution of dsRNA can be accomplished essentially asdescribed in Tabar et al. ((1998) Science 282:430-1)). Briefly,Hermaphrodite L4-stage C. elegans are washed twice in siliconized tubeswith approximately 1 ml M9 buffer (5 g/L NaCl, 11.32 g/L Na₂HPO₄.7H2O, 3g/L KH₂PO₄, 1 mM MgSO₄). In each wash, the worms are allowed to settlefor 10 minutes and most of the supernatant removed. Between five andtwenty worms in minimal volume (5-10 ul) are transferred to a freshsiliconized tube and an equal volume of specific dsRNA (resuspended insterile, RNase-free water) is added. The final concentration of dsRNA isgenerally between 0.1 and 3.0 mg/ml. Up to 10% (v/v) lipofectin(Gibco-BRL) may be added to the mix. The mixture is incubated for 10 to30 hours at a constant temperature between 15° and 23° C. The worms arethen transferred individually to NGM-agar plates containing a lawn of E.coli (such as strain OP50), incubated at constant temperature between15° and 23° C. and scored daily for phenotypes of the worms and theirprogeny for at least four consecutive days.

[0192] Screening Assays

[0193] Another embodiment of the present invention is a method ofidentifying a compound capable of altering (e.g., inhibiting orenhancing) the activity of fatty acid desaturase-like molecules. Thismethod, also referred to as a “screening assay,” herein, includes, butis not limited to, the following procedure: (i) contacting an isolatedfatty acid desaturase-like protein with a test inhibitory compound underconditions in which, in the absence of the test compound, the proteinhas fatty acid desaturase-like activity; and (ii) determining if thetest compound alters the fatty acid desaturase-like activity or altersthe ability of the fatty acid desaturase to regulate other polypeptidesor molecules e.g., the ability of the enzyme to desaturate fatty acids.Suitable inhibitors or activators that alter a nematode fatty aciddesaturase-like activity include compounds that interact directly with anematode fatty acid desaturase-like protein, perhaps but notnecessarily, in the active or binding site. They can also interact withother regions of the nematode fatty acid desaturase protein by bindingto regions outside of the active site or site responsible forregulation, for example, by allosteric interaction.

[0194] In one embodiment, an M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase-like polypeptide isexpressed in a yeast cell, for example in S. cerevisiae, as has beendescribed for a C. elegans FAT-2-like polypeptide (Peyo-Ndi (2000)Archives of Biochemistry and Biophysics 376:399-408). Overall fatty acidcomposition from wild-type and fatty acid desaturase-harboring yeast canthan be assessed using, for example, gas-chromotography-massspectrometry techniques (GC-MS). Optimally, an increase in Δ12unsaturated fatty acids would be concomitant with introduction of an M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like polypeptide into the yeast strain. Test compounds canthen be added to the yeast strain and fatty acid composition can bemeasured. A test compound that alters fatty acid composition,particularly decreases Δ12 unsaturated fatty acids in the yeast strainharboring the M. incognita, H. glycines, D. immitis, S. stercoralis orR. axei fatty acid desaturase-like polypeptides would be consideredcandidate compounds.

[0195] In one embodiment, an M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase-like polypeptide isexpressed in a eukaryotic or plant cell, for example in Chinese hamsterovary cells or rabbit skin cells. Overall fatty acid composition fromwild-type and fatty acid desaturase-harboring eukaryotic cells can thanbe assessed using, for example, gas chromatography-mass spectrometrytechniques (GC-MS). Optimally, an increase in Δ12 unsaturated fattyacids would be concomitant with introduction of an M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like polypeptide into the cells. Test compounds can then beadded to the eukaryotic cells and fatty acid composition can bemeasured. A test compound that alters fatty acid composition,particularly decreases Δ12 unsaturated fatty acids in the cellsharboring the M. incognita, H. glycines, D. immitis, S. stercoralis orR. axei fatty acid desaturase-like polypeptides would be consideredcandidate compounds.

[0196] Similarly, a M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase-like polypeptides can beexpressed in plants, for example in soybean, cotton, tobacco, potato, M.truncatula, and/or Arabidopsis. Plants used for transformation may havea functional Δ12 fatty acid desaturase gene or may be deficient in Δ12fatty acid desaturase activity. Relative amounts of fatty acids can bemeasured and compared. Compounds that alter fatty acid compositions,particularly those that decrease Δ12 unsaturated fatty acids, would becandidate compounds.

[0197] Compounds: A test compound can be a large or small molecule, forexample, an organic compound with a molecular weight of about 100 to10,000; 200 to 5,000; 200 to 2000; or 200 to 1,000 daltons. A testcompound can be any chemical compound, for example, a small organicmolecule, a carbohydrate, a lipid, an amino acid, a polypeptide, anucleoside, a nucleic acid, or a peptide nucleic acid. Small moleculesinclude, but are not limited to, metabolites, metabolic analogues,peptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e., includingheteroorganic and organometallic compounds). Compounds and componentsfor synthesis of compounds can be obtained from a commercial chemicalsupplier, e.g., Sigma-Aldrich Corp. (St. Louis, Mo.). The test compoundor compounds can be naturally occurring, synthetic, or both. A testcompound can be the only substance assayed by the method describedherein. Alternatively, a collection of test compounds can be assayedeither consecutively or concurrently by the methods described herein. Acompound may be an analog. Specifically, inhibitors may be analogs offatty acids, for example, cyclypropenoid analogs of linoleic acid. Epoxyfatty acids (vernolic acid), acetylenic fatty acids (crepenynic acid)and hydroxy fatty acids (ricinoleic acid) may be used as inhibitors.Analogs such as α-elostearic acid with conjugated double bonds may beacceptable inhibitors. These may be expressed in plants (e.g. a Δ12epoxygenase from C. palaestina produces vernolic acid in transgenicArabidopsis (Singh et. al., supra). Compounds can also act by allostericinhibition or by preventing the fatty acid desaturase from binding to,and thus, acting on its target, i.e., a fatty acid.

[0198] A high-throughput method can be used to screen large libraries ofchemicals. Such libraries of candidate compounds can be generated orpurchased, e.g., from Chembridge Corp. (San Diego, Calif.). Librariescan be designed to cover a diverse range of compounds. For example, alibrary can include 10,000, 50,000, or 100,000 or more unique compounds.Merely by way of illustration, a library can be constructed fromheterocycles including pyridines, indoles, quinolines, furans,pyrimidines, triazines, pyrroles, imidazoles, naphthalenes,benzimidazoles, piperidines, pyrazoles, benzoxazoles, pyrrolidines,thiphenes, thiazoles, benzothiazoles, and morpholines. It may becyclypropenoid analogs of linoleic acid, epoxy fatty acids (vernolicacid), acetylenic fatty acids (crepenynic acid), and/or hydroxy fattyacids (ricinoleic acid). Analogs such as a-elostearic acid withconjugated double bonds may be acceptable inhibitors. A library can bedesigned and synthesized to cover such classes of chemicals, e.g., asdescribed in DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909;Erb et al. (1994) Proc. Natl. Acad. Sci. USA, 91:11422; Zuckermann etal. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303;Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al.(1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J.Med. Chem. 37:1233.

[0199] Organism-based Assays: Organisms can be grown in microtiterplates, e.g., 6-well, 32-well, 64-well, 96-well, 384-well plates. In oneembodiment, the organism is a nematode. The nematodes can be geneticallymodified. Non-limiting examples of such modified nematodes include: 1)nematodes or nematode cells (M. incognita, H. glycines, D. immitis, S.stercoralis, R. axei, and/or C. elegans) having one or more fatty aciddesaturase-like genes inactivated (e.g., using RNA mediatedinterference); 2) nematodes or nematode cells expressing a heterologousfatty acid desaturase-like gene, e.g., a fatty acid desaturase-like genefrom another species; and 3) nematodes or nematode cells having one ormore endogenous fatty acid desaturase-like genes inactivated andexpressing a heterologous fatty acid desaturase-like gene, e.g., a M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like gene as described herein.

[0200] A plurality of candidate compounds, e.g., a combinatoriallibrary, can be screened.

[0201] The library can be provided in a format that is amenable forrobotic manipulation, e.g., in microtitre plates. Compounds can be addedto the wells of the microtiter plates. Following compound addition andincubation, viability and/or reproductive properties of the nematodes ornematode cells are monitored.

[0202] The compounds can also be pooled, and the pools tested. Positivepools are split for subsequent analysis. Regardless of the method,compounds that decrease the viability or reproductive ability ofnematodes, nematode cells, or progeny of the nematodes are consideredlead compounds.

[0203] In another embodiment, the compounds can be tested on amicroorganism, (e.g., a yeast and/or bacterium). For example, a M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase gene can be expressed in S. cerevisiae, as has been describedfor a C. elegans FAT-2 like polypeptide (Peyo-Ndi (2000) Archives ofBiochem and Biophysics 376: 399-408). The generation of such strains isroutine in the art. As described above for nematodes and nematode cells,the cell lines can be grown in microtitre plates, each well having adifferent candidate compound or pool of candidate compounds. Growth ismonitored during or after the assay to determine if the compound or poolof compounds is a modulator of a nematode fatty acid desaturase-likepolypeptide.

[0204] In another embodiment fatty acid composition of the organisms canbe determined, using, for example, GC-MS. Fatty acid methyl esters canbe formed by a transesterification reaction and extracted. GC-MS can beperformed using a Hewlet Packard 6890 gas chromatograph interfaced witha Hewlet Packard 5973 mass selective detector, for example.

[0205] Retention times of resulting methyl esters can be compared withthose of known samples (Cahoon, supra). In another embodiment, ethylesters can be extracted with hexane and analyzed by GLC through a 50 mby 0.32 mm CP-Wax58-CB fused silica column (Chrompack) (Singh, supra;Lee, supra). In another embodiment, if a M. incognita, H. glycines, D.immitis, S. stercoralis or R. axei fatty acid desaturase gene isexpressed in a microorganism (i.e., yeast), fatty acid methyl esters canbe prepared using methanolic HCl as described (Reed et al. (2000) PlantPhysiol. 122:715-720; Meesapyodsuk, (2000) Biochemistry 39:11948-11954).

[0206] In Vitro Activity Assays: The screening assay can be an in vitroactivity assay. For example, a nematode fatty acid desaturase-likepolypeptide can be purified as described above. The polypeptide can bedisposed in an assay container, e.g., a well of a microtitre plate alongwith an appropriate substrate. A candidate compound can be added to theassay container, and the fatty acid desaturase-like activity ismeasured. Optionally, the activity is compared to the activity measuredin a control container in which no candidate compound is disposed or inwhich an inert or non-functional compound is disposed. Fatty acidcomposition can be determined, using, for example, GC-MS. Fatty acidmethyl esters can be formed by a transesterification reaction andextracted. GC-MS can be performed using a Hewlet Packard 6890 gaschromatograph interfaced with a Hewlet Packard 5973 mass selectivedetector, for example. Retention times of resulting methyl esters can becompared with those of known samples (Cahoon, supra). In anotherembodiment, ethyl esters can be extracted with hexane and analyzed byGLC through a 50 m by 0.32 mm CP-Wax58-CB fused silica column(Chrompack) (Singh, supra; Lee, supra).

[0207] In Vitro Binding Assays: The screening assay can also be acell-free binding assay, e.g., an assay to identify compounds that binda nematode fatty acid desaturase-like polypeptide. For example, anematode fatty acid desaturase-like polypeptide can be purified andlabeled. The labeled polypeptide is contacted to beads; each bead has atag detectable by mass spectroscopy, and test compound, e.g., a compoundsynthesized by combinatorial chemical methods. Beads to which thelabeled polypeptide is bound are identified and analyzed by massspectroscopy. The beads can be generated using “split-and-pool”synthesis. The method can further include a second assay to determine ifthe compound alters the activity of the fatty acid desaturase-likepolypeptide.

[0208] Optimization of a Compound: Once a lead compound has beenidentified, standard principles of medicinal chemistry can be used toproduce derivatives of the compound. Derivatives can be screened forimproved pharmacological properties, for example, efficacy,pharmacokinetics, stability, solubility, and clearance. The moietiesresponsible for a compound's activity in the above-described assays canbe delineated by examination of structure-activity relationships (SAR)as is commonly practiced in the art. One can modify moieties on a leadcompound and measure the effects of the modification on the efficacy ofthe compound to thereby produce derivatives with increased potency. Foran example, see Nagarajan et al. (1988) J. Antibiot. 41:1430-8. Amodification can include N-acylation, amination, amidation, oxidation,reduction, alkylation, esterification, and hydroxylation. Furthermore,if the biochemical target of the lead compound is known or determined,the structure of the target and the lead compound can inform the designand optimization of derivatives. Molecular modeling software iscommercially available (e.g., Molecular Simulations, Inc.). “SAR byNMR,” as described in Shuker et al. (1996) Science 274:1531-4, can beused to design ligands with increased affinity, by joininglower-affinity ligands.

[0209] A preferred compound is one that interferes with the function ofa fatty acid desaturase-like polypeptide and that is not substantiallytoxic to plants, animals, or humans. By “not substantially toxic” it ismeant that the compound does not substantially affect the respectiveanimal, or human fatty acid desaturase proteins or fatty acid desaturaseactivity. Thus, particularly desirable inhibitors of M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase donot substantially inhibit fatty acid desaturase-like polypeptides orfatty acid desaturase activity of vertebrates, e.g., humans for example.Other desirable compounds do not substantially inhibit to fatty aciddesaturase activity of plants.

[0210] Standard pharmaceutical procedures can be used to assess thetoxicity and therapeutic efficacy of a modulator of a fatty aciddesaturase-like activity. The LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population can be measured in cell cultures, experimental plants(e.g., in laboratory or field studies), or experimental animals.Optionally, a therapeutic index can be determined which is expressed asthe ratio: LD50/ED50. High therapeutic indices are indicative of acompound being an effective fatty acid desaturase-like inhibitor, whilenot causing undue toxicity or side-effects to a subject (e.g., a hostplant or host animal).

[0211] Alternatively, the ability of a candidate compound to modulate anon-nematode fatty acid desaturase-like polypeptide is assayed, e.g., bya method described herein. For example, the inhibition constant of acandidate compound for a mammalian fatty acid desaturase-likepolypeptide can be measured and compared to the inhibition constant fora nematode fatty acid desaturase-like polypeptide.

[0212] The aforementioned analyses can be used to identify and/or designa modulator with specificity for nematode fatty acid desaturase-likepolypeptide over vertebrate or other animal (e.g., mammalian) fatty aciddesaturase-like polypeptides. Suitable nematodes to target are anynematodes with the fatty acid desaturase-like proteins or proteins thatcan be targeted by a compound that otherwise inhibits, reduces,activates, or generally affects the activity of nematode fatty aciddesaturase proteins.

[0213] Inhibitors of nematode fatty acid desaturase-like proteins canalso be used to identify fatty acid desaturase-like proteins in thenematode or other organisms using procedures known in the art, such asaffinity chromatography. For example, a specific antibody may be linkedto a resin and a nematode extract passed over the resin, allowing anyfatty acid desaturase-like proteins that bind the antibody to bind theresin. Subsequent biochemical techniques familiar to those skilled inthe art can be performed to purify and identify bound fatty aciddesaturase-like proteins.

[0214] Agricultural Compositions

[0215] A compound that is identified as a fatty acid desaturase-likepolypeptide inhibitor can be formulated as a composition that is appliedto plants, soil, or seeds in order to confer nematode resistance. Thecomposition can be prepared in a solution, e.g., an aqueous solution, ata concentration from about 0.005% to 10%, or about 0.01% to 1%, or about0.1% to 0.5% by weight. The solution can include an organic solvent,e.g., glycerol or ethanol. The composition can be formulated with one ormore agriculturally acceptable carriers. Agricultural carriers caninclude: clay, talc, bentonite, diatomaceous earth, kaolin, silica,benzene, xylene, toluene, kerosene, N-methylpyrrolidone, alcohols(methanol, ethanol, isopropanol, n-butanol, ethylene glycol, propyleneglycol, and the like), and ketones (acetone, methylethyl ketone,cyclohexanone, and the like). The formulation can optionally furtherinclude stabilizers, spreading agents, wetting extenders, dispersingagents, sticking agents, disintegrators, and other additives, and can beprepared as a liquid, a water-soluble solid (e.g., tablet, powder orgranule), or a paste.

[0216] Prior to application, the solution can be combined with anotherdesired composition such as another anthelmintic agent, germicide,fertilizer, plant growth regulator and the like. The solution may beapplied to the plant tissue, for example, by spraying, e.g., with anatomizer, by drenching, by pasting, or by manual application, e.g., witha sponge. The solution can also be distributed from an airborne source,e.g., an aircraft or other aerial object, e.g., a fixture mounted withan apparatus for spraying the solution, the fixture being of sufficientheight to distribute the solution to the desired plant tissues.Alternatively, the composition can be applied to plant tissue from avolatile or airborne source. The source is placed in the vicinity of theplant tissue and the composition is dispersed by diffusion through theatmosphere. The source and the plant tissue to be contacted can beenclosed in an incubator, growth chamber, or greenhouse, or can be insufficient proximity that they can be outdoors.

[0217] If the composition is distributed systemically thorough theplant, the composition can be applied to tissues other than the leaves,e.g., to the stems or roots. Thus, the composition can be distributed byirrigation. The composition can also be injected directly into roots orstems.

[0218] A skilled artisan would be able to determine an appropriatedosage for formulation of the active ingredient of the composition. Forexample, the ED50 can be determined as described above from experimentaldata. The data can be obtained by experimentally varying the dose of theactive ingredient to identify a dosage effective for killing a nematode,while not causing toxicity in the host plant or host animal (i.e.non-nematode animal).

[0219] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A purified polypeptide comprising an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO:
 8. 2. The purified polypeptide of claim 1, wherein the aminoacid sequence is at least 90% identical to the amino acid sequence ofSEQ ID NO:
 8. 3. The purified polypeptide of claim 2, wherein the aminoacid sequence is at least 95% identical to the amino acid sequence ofSEQ ID NO:
 8. 4. A purified polypeptide comprising the amino acidsequence of SEQ ID NO:
 8. 5. A purified polypeptide comprising an aminoacid sequence that is at least 80% identical to the amino acid sequenceof SEQ ID NO:
 9. 6. The purified polypeptide of claim 5, wherein theamino acid sequence is at least 90% identical to the amino acid sequenceof SEQ ID NO:
 9. 7. The purified polypeptide of claim 6, wherein theamino acid sequence is at least 95% identical to the amino acid sequenceof SEQ ID NO:
 9. 8. A purified polypeptide comprising the amino acidsequence of SEQ ID NO:
 9. 9. A purified polypeptide comprising an aminoacid sequence that is at least 80% identical to the amino acid sequenceof SEQ ID NO:
 10. 10. The purified polypeptide of claim 9, wherein theamino acid sequence is at least 90% identical to the amino acid sequenceof SEQ ID NO:
 10. 11. The purified polypeptide of claim 10, wherein theamino acid sequence is at least 95% identical to the amino acid sequenceof SEQ ID NO:
 10. 12. A purified polypeptide comprising the amino acidsequence of SEQ ID NO:
 10. 13. A purified polypeptide comprising anamino acid sequence that is at least 80% identical to the amino acidsequence of SEQ ID NO:
 11. 14. The purified polypeptide of claim 13,wherein the amino acid sequence is at least 90% identical to the aminoacid sequence of SEQ ID NO:
 11. 15. The purified polypeptide of claim14, wherein the amino acid sequence is at least 95% identical to theamino acid sequence of SEQ ID NO:
 11. 16. A purified polypeptidecomprising the amino acid sequence of SEQ ID NO:
 11. 17. A purifiedpolypeptide comprising an amino acid sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO:
 12. 18. The purifiedpolypeptide of claim 17, wherein the amino acid sequence is at least 90%identical to the amino acid sequence of SEQ ID NO:
 12. 19. The purifiedpolypeptide of claim 18, wherein the amino acid sequence is at least 95%identical to the amino acid sequence of SEQ ID NO:
 12. 20. A purifiedpolypeptide comprising the amino acid sequence of SEQ ID NO:
 12. 21. Apurified polypeptide comprising an amino acid sequence that is at least80% identical to the amino acid sequence of SEQ ID NO:
 13. 22. Thepurified polypeptide of claim 21, wherein the amino acid sequence is atleast 90% identical to the amino acid sequence of SEQ ID NO:
 13. 23. Thepurified polypeptide of claim 22, wherein the amino acid sequence is atleast 95% identical to the amino acid sequence of SEQ ID NO:
 13. 24. Apurified polypeptide comprising the amino acid sequence of SEQ ID NO:13.
 25. A purified polypeptide comprising an amino acid sequence that isat least 80% identical to the amino acid sequence of SEQ ID NO:
 14. 26.The purified polypeptide of claim 25, wherein the amino acid sequence isat least 90% identical to the amino acid sequence of SEQ ID NO:
 14. 27.The purified polypeptide of claim 26, wherein the amino acid sequence isat least 95% identical to the amino acid sequence of SEQ ID NO:
 14. 28.A purified polypeptide comprising the amino acid sequence of SEQ ID NO:14.
 29. An isolated nucleic acid encoding the polypeptide of claim 3 or4.
 30. An isolated nucleic acid encoding the polypeptide of claim 7 or8.
 31. An isolated nucleic acid encoding the polypeptide of claim 11 or12.
 32. An isolated nucleic acid encoding the polypeptide of claim 15 or16.
 33. An isolated nucleic acid encoding the polypeptide of claim 19 or20.
 34. An isolated nucleic acid encoding the polypeptide of claim 23 or24.
 35. An isolated nucleic acid encoding the polypeptide of claim 27 or28.
 36. An isolated nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:
 1. 37. An isolated nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:
 2. 38. An isolatednucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3.
 39. An isolated nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:
 4. 40. An isolated nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:
 5. 41. An isolatednucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:6.
 42. An isolated nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:
 7. 43. A vector comprising the nucleic acidmolecule of claim
 29. 44. A host cell harboring the isolated nucleicacid molecule of claim
 29. 45. A vector comprising the nucleic acidmolecule of claim
 30. 46. A host cell harboring the isolated nucleicacid molecule of claim
 30. 47. A vector comprising the nucleic acidmolecule of claim
 31. 48. A host cell harboring the isolated nucleicacid molecule of claim
 31. 49. A vector comprising the nucleic acidmolecule of claim
 32. 50. A host cell harboring the isolated nucleicacid molecule of claim
 32. 51. A vector comprising the nucleic acidmolecule of claim
 33. 52. A host cell harboring the isolated nucleicacid molecule of claim
 33. 53. A vector comprising the nucleic acidmolecule of claim
 34. 54. A host cell harboring the isolated nucleicacid molecule of claim
 34. 55. A vector comprising the nucleic acidmolecule of claim
 35. 56. A host cell harboring the isolated nucleicacid molecule of claim
 35. 57. A method comprising: (a) providing apolypeptide comprising the amino acid sequence of SEQ ID NO: 8; (b)contacting a test compound to the polypeptide; and (c) measuring thebinding of the test compound to the polypeptide.
 58. A methodcomprising: (a) providing a polypeptide comprising the amino acidsequence of SEQ ID NO: 9; (b) contacting a test compound to thepolypeptide; and (c) measuring the binding of the test compound to thepolypeptide.
 59. A method comprising: (a) providing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 10; (b) contacting atest compound to the polypeptide; and (c) measuring the binding of thetest compound to the polypeptide.
 60. A method comprising: (a) providinga polypeptide comprising the amino acid sequence of SEQ ID NO:11; (b)contacting a test compound to the polypeptide; and (c) measuring thebinding of the test compound to the polypeptide.
 61. A methodcomprising: (a) providing a polypeptide comprising the amino acidsequence of SEQ ID NO: 12; (b) contacting a test compound to thepolypeptide; and (c) measuring the binding of the test compound to thepolypeptide.
 62. A method comprising: (a) providing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 13; (b) contacting atest compound to the polypeptide; and (c) measuring the binding of thetest compound to the polypeptide.
 63. A method comprising: (a) providinga polypeptide comprising the amino acid sequence of SEQ ID NO: 14; (b)contacting a test compound to the polypeptide; and (c) measuring thebinding of the test compound to the polypeptide.
 64. The method of claim57, further comprising measuring fatty acid desaturase-like activity ofthe polypeptide.
 65. The method of claim 58, further comprisingmeasuring fatty acid desaturase-like activity of the polypeptide. 66.The method of claim 59, further comprising measuring fatty aciddesaturase-like activity of the polypeptide.
 67. The method of claim 60,further comprising measuring fatty acid desaturase-like activity of thepolypeptide.
 68. The method of claim 61, further comprising measuringfatty acid desaturase-like activity of the polypeptide.
 69. The methodof claim 62, further comprising measuring fatty acid desaturase-likeactivity of the polypeptide.
 70. The method of claim 63, furthercomprising measuring fatty acid desaturase-like activity of thepolypeptide.
 71. The method of claim 57, further comprising: (d)providing a second polypeptide, wherein the second polypeptide comprisesthe amino acid sequence of a mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring the binding of the test compoundto the second polypeptide.
 72. The method of claim 58, furthercomprising: (d) providing a second polypeptide, wherein the secondcomprises the amino acid sequence of a mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring the binding of the test compoundto the second polypeptide.
 73. The method of claim 59, furthercomprising: (d) providing a second polypeptide, wherein the secondcomprises the amino acid sequence of a mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring the binding of the test compoundto the second polypeptide.
 74. The method of claim 60, furthercomprising: (d) providing a second polypeptide, wherein the secondcomprises the amino acid sequence of a mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring the binding of the test compoundto the second polypeptide.
 75. The method of claim 61, furthercomprising: (d) providing a second polypeptide, wherein the secondcomprises the amino acid sequence of a mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring the binding of the test compoundto the second polypeptide.
 76. The method of claim 62, furthercomprising: (d) providing a second polypeptide, wherein the secondcomprises the amino acid sequence of a mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring the binding of the test compoundto the second polypeptide.
 77. The method of claim 63, furthercomprising: (d) providing a second polypeptide, wherein the secondcomprises the amino acid sequence of a mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring the binding of the test compoundto the second polypeptide.
 78. A method comprising: (a) providing apolypeptide comprising the amino acid sequence of SEQ ID NO: 8; (b)contacting a test compound to the polypeptide; and (c) measuring a fattyacid desaturase-like activity of the polypeptide, wherein a change infatty acid desaturase-like activity relative to the fatty aciddesaturase-like activity of the polypeptide in the absence of the testcompound is an indication that the test compound alters the activity ofthe polypeptide.
 79. A method comprising: (a) providing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 9; (b) contacting atest compound to the polypeptide; and (c) measuring a fatty aciddesaturase-like activity of the polypeptide, wherein a change in fattyacid desaturase-like activity relative to the fatty acid desaturase-likeactivity of the polypeptide in the absence of the test compound is anindication that the test compound alters the activity of thepolypeptide.
 80. A method comprising: (a) providing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 10; (b) contacting atest compound to the polypeptide; and (c) measuring a fatty aciddesaturase-like activity of the polypeptide, wherein a change in fattyacid desaturase-like activity relative to the fatty acid desaturase-likeactivity of the polypeptide in the absence of the test compound is anindication that the test compound alters the activity of thepolypeptide.
 81. A method comprising: (a) providing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 11; (b) contacting atest compound to the polypeptide; and (c) measuring a fatty aciddesaturase-like activity of the polypeptide, wherein a change in fattyacid desaturase-like activity relative to the fatty acid desaturase-likeactivity of the polypeptide in the absence of the test compound is anindication that the test compound alters the activity of thepolypeptide.
 82. A method comprising: (a) providing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 12; (b) contacting atest compound to the polypeptide; and (c) measuring a fatty aciddesaturase-like activity of the polypeptide, wherein a change in fattyacid desaturase-like activity relative to the fatty acid desaturase-likeactivity of the polypeptide in the absence of the test compound is anindication that the test compound alters the activity of thepolypeptide.
 83. A method comprising: (a) providing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 13; (b) contacting atest compound to the polypeptide; and (c) measuring a fatty aciddesaturase-like activity of the polypeptide, wherein a change in fattyacid desaturase-like activity relative to the fatty acid desaturase-likeactivity of the polypeptide in the absence of the test compound is anindication that the test compound alters the activity of thepolypeptide.
 84. A method comprising: (a) providing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 14; (b) contacting atest compound to the polypeptide; and (c) measuring a fatty aciddesaturase-like activity of the polypeptide, wherein a change in fattyacid desaturase-like activity relative to the fatty acid desaturase-likeactivity of the polypeptide in the absence of the test compound is anindication that the test compound alters the activity of thepolypeptide.
 85. The method of claim 78, further comprising the stepsof: (d) providing a second polypeptide, wherein the second polypeptidecomprises the amino acid sequence of a mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring an fatty acid desaturase-likeactivity of the second polypeptide.
 86. The method of claim 79, furthercomprising: (d) providing a second polypeptide, wherein the second fattyacid desaturase-like polypeptide is mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring a fatty acid desaturase-likeactivity of the second polypeptide.
 87. The method of claim 80, furthercomprising: (d) providing a second polypeptide, wherein the second fattyacid desaturase-like polypeptide is mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring a fatty acid desaturase-likeactivity of the second polypeptide.
 88. The method of claim 81, furthercomprising: (d) providing a second polypeptide, wherein the second fattyacid desaturase-like polypeptide is mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring a fatty acid desaturase-likeactivity of the second polypeptide.
 89. The method of claim 82, furthercomprising: (d) providing a second polypeptide, wherein the second fattyacid desaturase-like polypeptide is mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring a fatty acid desaturase-likeactivity of the second polypeptide.
 90. The method of claim 83, furthercomprising: (d) providing a second polypeptide, wherein the second fattyacid desaturase-like polypeptide is mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring a fatty acid desaturase-likeactivity of the second polypeptide.
 91. The method of claim 84, furthercomprising: (d) providing a second polypeptide, wherein the second fattyacid desaturase-like polypeptide is mammalian or plant fatty aciddesaturase-like polypeptide; (e) contacting the test compound to thesecond polypeptide; and (f) measuring a fatty acid desaturase-likeactivity of the second polypeptide.
 92. An antibody that bindsspecifically to a polypeptide selected from the group consisting of SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, and SEQ ID NO:
 14. 93. A method for identifying an inhibitorof a fatty acid desaturase-like polypeptide, comprising: (a) providing acell expressing a fatty acid desaturase-like polypeptide; (b) contactingthe cell with a test compound; (c) measuring the fatty aciddesaturase-like polypeptide activity of the cell, wherein a change infatty acid desaturase-like polypeptide activity of the cell relative tothe fatty acid desaturase-like polypeptide activity of the cell in theabsence of the test compound is an indication that the test compoundalters the activity of the fatty acid desaturase-like polypeptide.
 94. Amethod for identifying an inhibitor of a fatty acid desaturase-likepolypeptide, comprising: (a) providing a nematode expressing a fattyacid desaturase-like polypeptide; (b) contacting the nematode with atest compound; (c) measuring the fatty acid desaturase-like polypeptideactivity of the nematode, wherein a change in fatty acid desaturase-likepolypeptide activity of the nematode relative to the fatty aciddesaturase-like polypeptide activity of the nematode in the absence ofthe test compound is an indication that the test compound alters theactivity of the fatty acid desaturase-like polypeptide.
 95. A method foridentifying an inhibitor of a fatty acid desaturase-like polypeptide,comprising: (a) providing a cell expressing a fatty acid desaturase-likepolypeptide; (b) contacting the cell with a test compound; (c) measuringthe viability of the cell in the presence of the test compound; and (d)comparing the viability of the cell in the presence of the test compoundto the viability of the cell in the presence of the test compound and aproduct the fatty acid desaturase-like polypeptide, wherein greaterviability in the presence of the test compound and the product comparedto viability in the presence of the test compound is an indication thatthe test compound alters the activity of the fatty acid desaturase-likepolypeptide.
 96. A method for identifying an inhibitor of a fatty aciddesaturase-like polypeptide, comprising: (a) providing a nematodeexpressing a fatty acid desaturase-like polypeptide; (b) contacting thenematode with a test compound; (c) measuring the viability of the cellin the presence of the test compound; and (d) comparing the viability orfecundity of the nematode in the presence of the test compound to theviability or fecundity of the nematode in the presence of the testcompound and a product the fatty acid desaturase-like polypeptide,wherein greater viability or fecundity in the presence of the testcompound and the product compared to viability or fecundity in thepresence of the test compound is an indication that the test compoundalters the activity of the fatty acid desaturase-like polypeptide. 97.The method of claim 95 or claim 96 wherein the product is linoleic acid.98. A method for reducing the viability, growth, or fecundity of anematode, the method comprising exposing the nematode to an agent thatinhibits the activity of a fatty acid desaturase-like polypeptide. 99.The method of claim 98 wherein the fatty acid desaturase-likepolypeptide is a Δ12 fatty acid desaturase.
 100. An inhibitor identifiedby the method of any one of claims 93-97.
 101. A method for protecting aplant from a nematode infection, the method comprising applying to theplant, its environment or to seeds of the plant an inhibitor of anematode fatty acid desaturase-like polypeptide.
 102. A method forprotecting a mammal from a nematode infection, the method comprisingadministering to the mammal an inhibitor of a nematode fatty aciddesaturase-like polypeptide.
 103. The method of claim 101 or 102 whereinthe fatty acid desaturase-like polypeptide is a Δ12 fatty aciddesaturase.
 104. The method of claim 101 wherein the inhibitor does notsignificantly inhibit the activity of a fatty acid desaturase-likepolypeptide expressed by the plant.
 105. The method of claim 102 whereinthe inhibitor does not significantly inhibit the activity of a fattyacid desaturase-like polypeptide expressed by the mammal.
 106. Acomposition suitable for administration to plants comprising aninhibitor of a nematode fatty acid desaturase.